U.S. patent application number 14/516753 was filed with the patent office on 2015-02-26 for anti-cd40 antibody mutants.
This patent application is currently assigned to KYOWA HAKKO KIRIN CO., LTD.. The applicant listed for this patent is KYOWA HAKKO KIRIN CO., LTD.. Invention is credited to YOSHINORI KITAGAWA, AKI MATSUSHIMA, TORU MIURA, NOBUAKI TAKAHASHI.
Application Number | 20150057437 14/516753 |
Document ID | / |
Family ID | 34736433 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150057437 |
Kind Code |
A1 |
TAKAHASHI; NOBUAKI ; et
al. |
February 26, 2015 |
ANTI-CD40 ANTIBODY MUTANTS
Abstract
A mutant of a potentially therapeutic anti-CD40 antibody is
provided which mutant has reduced ADCC and CDC activities designed
to be optimized as a pharmaceutical agent. A mutant of an agonistic
anti-CD40 antibody, comprising mutation and/or substitution of at
least one amino acid in the constant region to reduce the ADCC
and/or CDC activities therein, and a mutant of an antagonistic
anti-CD40 antibody, comprising at least one mutation or
substitution in the constant region to reduce the ADCC and/or CDC
activities therein, both mutants having at least a hinge region
derived from a human IgG2.
Inventors: |
TAKAHASHI; NOBUAKI; (TOKYO,
JP) ; MIURA; TORU; (TOKYO, JP) ; KITAGAWA;
YOSHINORI; (TOKYO, JP) ; MATSUSHIMA; AKI;
(TOKYO, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KYOWA HAKKO KIRIN CO., LTD. |
TOKYO |
|
JP |
|
|
Assignee: |
KYOWA HAKKO KIRIN CO., LTD.
TOKYO
JP
|
Family ID: |
34736433 |
Appl. No.: |
14/516753 |
Filed: |
October 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14254413 |
Apr 16, 2014 |
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14516753 |
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14017789 |
Sep 4, 2013 |
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14254413 |
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10584345 |
Feb 26, 2007 |
8568725 |
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PCT/JP2004/019750 |
Dec 24, 2004 |
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14017789 |
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Current U.S.
Class: |
530/387.9 ;
435/69.6 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/732 20130101; A61P 37/00 20180101; A61P 37/06 20180101;
A61P 35/00 20180101; C07K 2317/56 20130101; C07K 2317/734 20130101;
C07K 2317/34 20130101; A61P 37/08 20180101; C07K 16/2878 20130101;
A61P 7/04 20180101; C07K 2317/53 20130101; A61P 7/00 20180101; A61P
31/00 20180101; C07K 2317/73 20130101; C07K 2317/52 20130101 |
Class at
Publication: |
530/387.9 ;
435/69.6 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 25, 2003 |
JP |
2003-431408 |
Claims
1. An antibody prepared by a process comprising: (i) culturing an
animal cell containing (a) a nucleic acid that encodes a heavy
chain consisting of an amino acid sequence comprising from Q at
position 27 to K at position 474 of SEQ ID NO: 140 and (b) a
nucleic acid that encodes an light chain consisting of an amino
acid sequence comprising from A at position 23 to C at position 235
of SEQ ID NO: 142 to produce an antibody that binds human CD40; and
(ii) purifying the antibody.
2. The antibody of claim 1, wherein the heavy chain is produced by
removing the signal sequence from the polypeptide represented by
SEQ ID NO: 140.
3. The antibody of claim 1, wherein the heavy chain consists of the
amino acid sequence set forth in SEQ ID NO: 46.
4. The antibody of claim 1, wherein the heavy chain comprises a
variable region consisting of amino acid residues 27 to 147 of SEQ
ID NO: 46.
5. The antibody of claim 1, wherein the light chain is produced by
removing the signal sequence from the polypeptide represented by
SEQ ID NO: 142.
6. The antibody of claim 1, wherein the light chain consists of the
amino acid sequence set forth in SEQ ID NO: 48.
7. The antibody of claim 1, wherein the light chain comprises a
variable region consisting of amino acid residues 23 to 128 of SEQ
ID NO: 48.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/254,413, filed Apr. 4, 2014, which is a continuation of U.S.
application Ser. No. 14/017,789, filed Sep. 4, 2013, which is a
continuation of U.S. application Ser. No. 10/584,345, filed Feb.
26, 2007, issued as U.S. Pat. No. 8,568,725 on Oct. 29, 2013, and
which is a U.S. National Phase of International Application
PCT/JP2004/019750, filed Dec. 24, 2004, which was published on Jul.
14, 2005, as WO 2005/063981, which claims the benefit of JP
Application No. 2003-431408, filed Dec. 25, 2003, all of which are
incorporated herein by reference in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to an anti-CD40 antibody which
recognizes CD40 which is a type of cell membrane molecules
associated with immunity. Further, the present invention relates to
an antibody with a mutation in the constant region of the human
antibody or with a subclass having its portion substituted in order
to decrease an ADCC and/or CDC activity, while keeping an agonistic
or antagonistic activity.
BACKGROUND ART
1. CD40
[0003] CD40 is an antigen having a molecular weight of 50 kDa which
is present on the surface of cell membrane, and expressed in B
cells, dendritic cells (DCs), some types of cancer cells, and
thymic epithelial cells. CD40 is known to play an important role in
proliferation and differentiation of B cells and DCs. CD40 was
identified as an antigen expressed on the surface of human B cells
(E. A. Clark et al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986; and
I. Stamenkovic et al., EMBO J. 8: 1403, 1989) and has been
considered as a member of the TNF receptor family which includes
low-affinity NGF receptors, TNF receptors, CD27, OX40 and CD30.
Ligands (CD40Ls) to human and murine CD40s have been recently
cloned and found to be membrane proteins type II and expressed in
activated CD4+T cells. CD40L has been also found to introduce
strong signals for activation into human or murine B cells.
[0004] In dendritic cells, CD40 has been observed to be more highly
expressed than in B cells, and it has become clear that CD40 plays
an important role in dendritic cells. Binding of CD40 to CD40L
activates antigen presenting cells (APCs), that is, expresses
costimulator molecules such as CD80 (B7-1) and CD86 (B7-2) or
enhances production of IL-2 (Caux, C., et al.: Activation of human
dendritic cells through CD40 cross-linking. J. Exp. Med., 180:
1263, 1994; and Shu, U., et al.: Activated T cells induce
interleukin-12 production by monocyte via CD40-CD40 ligand
interaction. Eur. J. Immunol, 25: 1125, 1995). Dendritic cells have
a strong antigen-presenting capacity and a strong capacity to
activate helper T (Th) cells. Dendritic cells are also believed to
control differentiation of naive Th cells into Th1 or Th2 cells.
When peripheral blood monocytes, which are myeloid dendritic cells,
are cultured in the presence of GM-CSF and IL-4, and matured by
CD40L, the resulting matured dendritic cells (DC1) can produce
IL-12 in vitro, and stimulate and activate allogeneic naive Th
cells to induce IFN.gamma.-producing T cells (i.e., to promote
their differentiation into Th1). This action is inhibited by
anti-IL-12 antibody and hence may be effected via IL-12. On the
other hand, when plasmacytoid T cells, which are present in
lymphoid T regions and peripheral blood, are cultured in the
presence of IL-3 and CD40 ligand, the resulting lymphoid dendritic
cells (DC2) are shown to be unable to produce IL-12, and stimulate
and activate allogeneic naive Th cells to induce IL-4-producing T
cells, which indicates promotion of their differentiation into Th2.
It is believed that Th1 cells are involved in activation of
cellular immunity, while Th2 cells are associated with enhancement
of humoral immunity as well as restriction of cellular immunity.
When cytotoxic T cells (CTL) are activated with the help of Th1
cells, they may eliminate pathogens (a number of types of virus,
listeria, tuberculosis bacteria, toxoplasma protozoa, etc.) growing
in the cytoplasm and tumor cells.
[0005] Monoclonal anti-CD40 antibodies, which recognize CD40
expressed on the membrane surface, have been demonstrated to have
different biological activities to B cells. Monoclonal anti-CD40
antibodies are generally classified into agonistic or antagonistic
antibodies against the interaction between CD40 and CD40L.
2. Agonistic Antibodies
[0006] Agonistic antibodies are known to activate B cells. For
instance, the anti-CD40 antibodies are reported to induce cell
adhesion (Barrett et al., J. Immunol. 146: 1722, 1991; and Gordon
et al., J. Immunol. 140: 1425, 1998), increase cell size (Gordon et
al., J. Immunol. 140: 1425, 1998; and Valle et al., Eur. J. Immunol
19: 1463, 1989), induce cell division of B cells activated only by
an anti-IgM antibody, anti-CD20 antibody or phorbol ester (Clark
and Ledbetter, Proc. Natl. Acad. Sci. USA 83: 4494, 1986; Gordon et
al., LEUCOCYTE TYPING III. A. J. McMicheal ed. Oxford University
Press. Oxford. p. 426; and Paulie et al., J. Immunol. 142: 590,
1989), induce cell division of B cells in the presence of IL4
(Valle et al., Eur. J. Immunol. 19: 1463, 1989; and Gordon et al.,
Eur. J. Immunol. 17: 1535, 1987), induce expression of IgE by
cultured cells stimulated with IL-4 and deprived of T cells (Jabara
et al., J. Exp. Med. 172: 1861, 1990; and Gascan et al., J.
Immunol. 147: 8, 1991), induce expression of IgG and IgM by those
cultured cells (Gascan et al., J. Immunol. 147: 8, 1991), secrete
soluble CD23/FceRII from cells via IL-4 (Gordon and Guy, Immunol.
Today 8: 39, 1987; and Cairns et al., Eur. J. Immunol. 18: 349,
1988), enhance expression of soluble CD23/FceRII on the cells via
IL4 (Challa, A., Allergy, 54: 576, 1999), and promote IL-6
production (Clark and Shu, J. Immunol. 145: 1400, 1990).
Furthermore, it is reported that addition of IL-4 and an anti-CD40
antibody to human primary culture B cells in the presence of
CDw32+adhesive cells led to establishment of cloned B cells derived
therefrom (Bancherauet et al., Science 241: 70, 1991), and
apoptosis of germinal center cells was inhibited through CD40
irrespective of whether its antigen receptor was active or inactive
(Liu et al., Nature 342: 929, 1989). As described above, CD40 has
been identified as antigen expressed on the surface of human B
cells, and consequently, most of the isolated antibodies have been
evaluated, as an index, mainly using their induction potency for
proliferation and/or differentiation of human B cells, or their
induction activity for cell death of cancer cells (Katira, A. et
al., LEUKOCYTE TYPING V. S. F. Schlossossman, et. al. eds. p. 547.
Oxford University Press. Oxford; W. C. Flansow et. al., LEUKOCYTE
TYPING V. S. F. Schlossossman, et. al. eds. p. 555. Oxford
University Press. Oxford; and J. D. Pound et. al., International
Immunology, 11: 11, 1999).
[0007] The anti-CD40 antibody has been demonstrated to mature DC
(Z. H. Zhou et. al., Hybridoma, 18: 471, 1999). Furthermore, the
role of CD4 T cells in priming antigen-specific CD8 T cells was
reported to be in activation of DC via CD40-CD40L signaling, and
the anti-CD40 monoclonal antibody (mAb) has been found to be able
to replace CD40 helper T cells in activation of dendritic cells
(DC) (Shoenberger, S. P., et. al.: T-cell help for cytotoxic T
lymphocytes is mediated by CD40-CD40L interactions. Nature, 480,
1998). Also, administration of an anti-CD40 antibody in mice has
been found to be able to protect the animal body from
CD40-expressing tumor cells as well as CD40-non-expressing tumor
cells (French, R. R., et. al.: CD40 antibody evokes a cytotoxic
T-cell response that eradicates lymphoma and bypasses T-cell help.
Nature Medicine, 5, 1999).
[0008] Agonistic anti-CD40 antibodies are expected to be effective
for treatment of infectious diseases, due to bacteria, virus, etc.,
cancer and others, based on their functions described above.
Anti-CD40 antibodies with superior agonistic activities are
described in WO 02/088186. The representative examples of those
agonistic antibodies are KM341-1-19 and 2105 antibodies. The
hybridoma KM341-1-19 producing the KM341-1-19 antibody and the
hybridoma 2105 producing the 2105 antibody were submitted on 27
Sep. 2001 and 17 Apr. 2002, respectively, for international deposit
under the Budapest Treaty, to International Patent Organisms
Depositary, National Institute of Advanced Industrial Science and
Technology (central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan).
Their accession numbers are FERM BP-7759 (KM341-1-19) and FERM
BP-8024 (2105).
3. Antagonistic Antibodies
[0009] Taking it in consideration, on the other hand, that CD40
plays an important role in immunologic responses, as
aforementioned, it is expected that inhibition of binding of CD40
to its ligands would lead to development of therapeutic agents for
immune suppression in organ transplantation and autoimmune
diseases. Sawada, Hase and others have reported that the peripheral
blood of patients suffering from Crohn's disease has a higher
percentage of monocytes highly expressing CD40. However, such
antibodies have not been well known yet as inhibit binding of CD40
to its ligands. Those inhibitory antibodies would be useful in
functional analysis of CD40 and treatment of diseases requiring
activation of CD40. Inhibitory antibodies to CD40 ligands are also
suggested to be effective against diseases involving binding of
CD40 to the CD40 ligands. However, CD40L was reported to be
expressed in activated platelets (V. Henn et al., Nature 391: 591,
1998), and if an anti-CD40L antibody is used as a therapeutic
agent, thrombus formation may occur reportedly (T. Kawai et al.,
Nat. Med. 6: 114, 2000). From this point of view, antibodies to
CD40 are expected to be safer rather than anti-CD40L antibodies as
therapeutic antibody agent to inhibit binding of CD40 to its
ligands. Anti-CD40 antibodies would be required to inhibit binding
of CD40L to CD40 and still not activate CD40 in themselves.
[0010] Such antagonistic anti-CD40 antibodies may be used for
treatment of autoimmune diseases and suppression of immunologic
rejections in transplantation of organs, bone marrow, etc., in view
of their functions described above. Anti-CD40 antibodies with
superior antagonistic activities are described in WO 02/088186. The
representative example of those antagonistic antibodies is 4D11
antibody. The hybridoma 4D11 producing the 4D11 antibody was
submitted on 27 Sep. 2001 for international deposit under the
Budapest Treaty, to International Patent Organisms Depositary,
National Institute of Advanced Industrial Science and Technology
(central 6, 1-1, Higashi 1, Tsukuba, Ibaraki, Japan). The accession
number is FERM BP-7758.
[0011] Patent Document 1 WO 02/088186
DISCLOSURE OF THE INVENTION
[0012] The object of the present invention is to create mutants
from the potentially therapeutic anti-CD40 antibodies disclosed in
WO 02/088186, which mutants are designed optimally as
pharmaceutical agent.
[0013] As a result of extensive and intensive research, the present
inventors have successfully created novel mutants of the agonistic
or antagonistic antibodies, which mutants may have a higher
therapeutic effect against diseases than known anti-CD40
antibodies, and completed the present invention based thereon. The
basic idea on modification of the anti-CD40 antibodies according to
the present invention will be described in detail below.
[0014] The present specification shall encompass the description in
the specification and/or drawings of JP Patent Publication (Kokai)
No. 2003-431408 which is the basis for the priority of the present
application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1A-1 shows binding site peptides (SEQ ID NOs: 49-88)
prepared based on the CD40 sequence, to which the anti-CD40
agonistic antibodies bind;
[0016] FIG. 1A-2 shows binding site peptides (SEQ ID NOs: 89-130)
prepared based on the CD40 sequence, to which the anti-CD40
agonistic antibodies bind (a continuation to FIG. 1A-1);
[0017] FIG. 1B-1 shows binding site peptides (SEQ ID NOs: 49-88)
prepared based on the CD40 sequence, to which the anti-CD40
antagonistic antibodies bind;
[0018] FIG. 1B-2 shows binding site peptides (SEQ ID NOs: 89-130)
prepared based on the CD40 sequence, to which the anti-CD40
antagonistic antibodies bind (a continuation to FIG. 1B-1);
[0019] FIG. 2A illustrates binding of the anti-CD40 antibodies to
the CD40 mutant;
[0020] FIG. 2B illustrates binding of the anti-CD40 antibodies to
the CD40 mutant;
[0021] FIG. 2C illustrates binding of the anti-CD40 antibodies to
the CD40 mutant;
[0022] FIG. 3A shows diagrams indicating that the KM341-1-19
antibody having a P331S mutation is as active as the original
KM341-1-19 antibody with respect to binding to Ramos cells;
[0023] FIG. 3B shows diagrams indicating that the KM341-1-19
antibody having a P331S mutation is as active as the original
KM341-1-19 antibody with respect to enhancement of CD95 expression
of Ramos cells;
[0024] FIG. 4A shows a diagram indicating that the KM341-1-19
antibody having a P331S mutation has a lower CDC activity via the
rabbit complement;
[0025] FIG. 4B shows a diagram indicating that the G2/4 antibody
has a lower complement activity when the human complement is
used;
[0026] FIG. 5A-1 shows diagrams indicating that conversion of the
subclass of the 2105 antibody from IgG2 into different subclasses
has no effect on its binding to Ramos cells;
[0027] FIG. 5A-2 shows diagrams indicating that conversion of the
subclass of the KM341-1-19 antibody from IgG2 into different
subclasses has no effect on its binding to Ramos cells;
[0028] FIG. 5B-1 shows diagrams indicating that conversion of the
subclass of the 2105 antibody from IgG2 into different subclasses
lowers an activity to enhance CD95 expression of Ramos cells;
[0029] FIG. 5B-2 shows diagrams indicating that conversion of the
subclass of the KM341-1-19 antibody from IgG2 into different
subclasses lowers an activity to enhance CD95 expression of Ramos
cells;
[0030] FIG. 6A-1 shows diagrams indicating that the binding
capacity of the KM341-1-19 antibodies to Ramos cells is independent
of the varying structure of the hinge region;
[0031] FIG. 6A-2 shows diagrams indicating that the binding
capacity of the 2105 antibodies to Ramos cells is independent of
the varying structure of the hinge region;
[0032] FIG. 6B-1 shows diagrams indicating that the upper and
middle hinges of the hinge region are important for the activity of
the KM341-1-19 antibodies to enhance CD95 expression of Ramos
cells;
[0033] FIG. 6B-2 shows diagrams indicating that the upper and
middle hinges of the hinge region are important for the activity of
the 2105 antibodies to enhance CD95 expression of Ramos cells;
[0034] FIG. 7A shows diagrams indicating that conversion of the
subclass of the F72 antibody to IgG2 has no effect on its binding
to Ramos cells;
[0035] FIG. 7B shows diagrams indicating that conversion of the
subclass of the F72 antibody to IgG2 raises an activity to enhance
CD95 expression of Ramos cells;
[0036] FIG. 8A shows diagrams indicating that conversion of the
subclass of the 4D11 antibody from IgG1 to IgG4 has no effect on
its binding to Ramos cells;
[0037] FIG. 8B shows diagrams indicating that conversion of the
subclass of the 4D11 antibody from IgG1 to IgG4 inhibits
enhancement by the CD40Ligand of CD95 expression of Ramos cells, at
the same extent as otherwise;
[0038] FIG. 9 shows a diagram indicating that conversion of the
subclass of the 4D11 antibody from IgG1 to IgG4 or IgG4PE lowers
the ADCC activity;
[0039] FIG. 10 shows a diagram indicating that conversion of the
subclass of the 4D11 antibody from IgG1 to IgG4P lowers the CDC
activity;
[0040] FIG. 11 illustrates a variation in number of B cells in the
blood (B220-positive cells among the peripheral blood lymphocytes)
over time after 4D11G1, 4D11G4P or 4D11G4PE was administered into
human CD40-transgenic mice;
[0041] FIG. 12A illustrates a higher expression of CD23 of splenic
B cells (CD23-positive cells among the splenic B cells) after each
anti-CD40 antibody was administered into human CD40-transgenic
mice;
[0042] FIG. 12B illustrates a higher expression of CD86 of splenic
B cells (CD86-positive cells among the splenic B cells) after each
anti-CD40 antibody was administered into human CD40-transgenic
mice;
[0043] FIG. 12C illustrates a higher expression of CD95 of splenic
B cells (CD95-positive cells among the splenic B cells) after each
anti-CD40 antibody was administered into human CD40-transgenic
mice;
[0044] FIG. 13A illustrates the suppressive activity of the
antigen-specific antibody (IgG1) production by 4D11 and 281-1-10 in
human CD40-transgenic mice;
[0045] FIG. 13B illustrates the suppressive activity of the
antigen-specific antibody (IgM) production by 4D11 and 281-1-10 in
human CD40-transgenic mice;
[0046] FIG. 14A illustrates the numbers of B cells in the blood
(B220-positive cells among the peripheral blood lymphocytes) during
the suppression assay of the antigen-specific antibody producing
activity;
[0047] FIG. 14B illustrates the numbers of splenic B cells
(B220-positive cells among the splenic lymphocytes) during the
suppression assay of the antigen-specific antibody producing
activity;
[0048] FIG. 15 illustrates a variation in number of B cells in the
blood (B220-positive cells among the peripheral blood lymphocytes)
over time after 4D11G4P or 4D11G4PE was administered at a dose of
30 mg/kg into cynomolgus monkeys;
[0049] FIG. 16 illustrates blood IL-12 levels during the assay
shown in FIG. 15;
[0050] FIG. 17 shows the suppressive effect of 4D11G4PE on the
simian DTH (delayed-type hypersensitivity in male cynomolgus
monkeys);
[0051] FIG. 18 shows the titers of the anti tetanus toxin IgG
during the assay with the results shown in FIG. 17;
[0052] FIG. 19 shows the titers of the anti tetanus toxin IgM
during the assay with the results shown in FIG. 17;
[0053] FIG. 20A illustrates the respective influences of 4D11G4PE
and 5C8 (anti-CD40Ligand antibody) on platelet aggregation;
[0054] FIG. 20B shows the respective influences of 4D11G4PE and 5C8
(anti-CD40Ligand antibody) on platelet aggregation;
[0055] FIG. 21 illustrates a variation in oligomer content of
4D11G4P, 4D11G4PE, 4D11G2Ser or 4D11G4/2/4 over time after it was
incubated at pH 2.7 and 37.degree. C.;
[0056] FIG. 22 illustrates suppression of rejection of skin grafts
by the anti-CD40 antagonistic antibody;
[0057] FIG. 23 illustrates the volume change of the tumor over time
from cell implantation, in a case where 341G2Ser was administered
to tumor bearing mice with Ramos cells implanted therein;
[0058] FIG. 24 illustrates the volume change of the tumor over time
from cell implantation, in a case where 341G2Ser was administered
to tumor bearing mice with T24 cells implanted therein;
[0059] FIG. 25 illustrates the volume change of the tumor over time
from cell implantation, in a case where 341G2Ser was administered
to tumor bearing mice with Hs 766T cells implanted therein; and
[0060] FIG. 26 illustrates the volume change of the tumor over time
from cell implantation, in a case where 341G2Ser was administered
to tumor bearing mice with Capan-2 cells implanted therein.
BEST MODE FOR CARRYING OUT THE INVENTION
1. Modification of Agonistic Antibodies
[0061] Antibodies are essentially molecules that function to
protect living bodies against foreign bodies, such as
microorganisms and viruses, and cancer, and hence they can kill and
eliminate such cells binding to themselves. The lethal activity is
composed of two different activities, called Antibody-Dependent
Cellular Cytotoxicity (abbreviated as ADCC hereinafter) and
Complement-Dependent Cytotoxicity (abbreviated as CDC
hereinafter).
[0062] ADCC refers to a type of cytotoxicity induced by activation
of macrophages, NK cells, neutrophil cells, etc., which recognize
target cells by binding to the constant region of the antibody via
Fc receptors expressed on their surface. In contrast, CDC refers to
a type of cytotoxicity induced by activation of a complement system
which occurs through binding of an antibody to an antigen. These
activities are known to vary depending on a subclass of the
antibody, which has been found to be due to a structural difference
in the constant region of antibodies (Charles A. Janeway et al.,
Immunology, 1997, Current Biology Ltd./Garland Publishing
Inc.).
[0063] Anti-CD40 agonistic antibodies will be more preferable as
therapeutic agent if they have not the activities of ADCC and/or
CDC which may induce cell death of CD40-expressing cells, in terms
of mechanism of immunoactive action. If CD40-expressing cells are
injured by ADCC and/or CDC activities, immunosuppression may occur
rather than desired immunoactivation, resulting in exacerbation of
the disease. In addition, patients suffering from infectious
diseases may have higher ADCC and/or CDC activities. Therefore,
when such antibodies are applied to infectious diseases, it is
necessary to evaluate them for safety more carefully, for example,
using more active rabbit complements than those present in healthy
human serum or peripheral blood which could not be effective to
detect the above activities in this situation. Accordingly, mutants
and recombinants were created which had no activity of ADCC or CDC
and examined for their activity.
[0064] Since ADCC and/or CDC activities are known to vary depending
on a subclass of the antibody of interest, conversion of the
subclass may reduce ADCC and/or CDC activities. For the human IgG
subclasses, for example, IgG4 is generally known to be a subclass
with low activities of both ADCC and CDC, and it is reported that
IgG2 is CDC active but poorly active in ADCC, while IgG1 is highly
active in both ADCC and CDC (Charles A. Janeway et al., Immunology,
1997, Current Biology Ltd./Garland Publishing Inc.). Selection of a
particular subclass by taking advantage of the above
characteristics may create a less cytotoxic antibody from the
original antibody. A combination of a specific subclass of antibody
with such a point mutation as described below may create an
antibody with a desired activity. Further, reduction in ADCC and/or
CDC activities of an antibody is reported to be attained by
incorporation of a mutation into its constant region. For instance,
L235, D265, D270, K322, P331, and P329 (each alphabetical letter
denotes an amino acid by the single-letter notation, and each
number denotes an EU index proposed by Kabat et al. (Kabat et. al.,
Sequences of proteins of Immunological Interest, 1991 Fifth
edition); such symbols will be used hereinafter.) may play an
important role in complement activation by human IgG, and
substitution of one of those sites by another amino acid may reduce
the CDC activity. Esohe E. Idusogie et. al. J. Immunol. 2000,
164:4178-4184, Yuanyuan Xu et. al. J. Biol. Chem. 1994,
269:3469-3474, Brekke, 0. H. et. al. Eur. J. Immunol. 1994,
24:2542, Morgan, A., et. al., Immunology 1995, 86:319, Lund, J.,
et. al:, J. Immunol., 1996, 157:4963, Tao, M. H., et. al., J. Exp.
Med. 1993, 178:661). Specifically, substitution of D270, K322,
P329, or P331 by A may reduce the CDC activity. Substitution of
P331 by S or G may also induce the same thing.
[0065] It is believed that Glu233-Ser239, Gly316-Lys338,
Lys274-Arg301, Tyr407-Arg416, Asn297, Glu318, Leu234-Ser239,
Asp265-Glu269, Asn297-Thr299 and Ala327-Ile332 take a part in
binding of IgG to FcR (Duncan, A. R., Woof, J. M., Partridge, L.
J., Burton, D. R., and Winter, G. (1988) Nature 332, 563-564,
Gessner, J. E., Heiken, H., Tamm, A., and Schmidt, R. E. (1998)
Ann. Hematol. 76, 231-248, Gavin, A., Hulett, M, and Hogarth, P. M.
(1998) in The Immunoglobulin Receptors and Their Physiological and
Pathological Roles in Immunity (van de Winkel, J. G. J., and
Hogarth, P. M., eds), pp. 11-35, Kluwer Academic Publishers Group,
Dordrecht, The Netherlands, Sautes, C. (1997) in Cell-mediated
Effects of Immunoglobulins (Fridman, W. H., and Sautes, C., eds),
pp. 29-66, R. G. Landes Co., Austin, Tex., Da'ron, M. (1997) Annu.
Rev. Immunol. 15, 203-234, Canfield, S. M., and Morrison, S. L.
(1991) J. Exp. Med. 173, 1483-1491, Chappel, M. S., Isenman, D. E.,
Everett, M., Xu, Y.-Y., Dorrington, K. J., and Klein, M. H. (1991)
Proc. Natl. Acad. Sci. U.S.A. 88, 9036-9040, Woof, J. M.,
Partridge, L. J., Jefferis, R., and Burton, D. R. (1986) Mol.
Immunol. 23, 319-330, Wines, B. D., Powell, M. S., Parren, P. W. H.
I., Barnes, N., and Hogarth, P. M. (2000) J. Imunol. 164,
5313-5318), and thus incorporation of a mutation into one of these
regions may reduce the ADCC activity. Specifically, substitution of
L235 by E or G237 by A can reduce binding of IgG to FcR.
[0066] The antibody according to the present invention has at least
one mutation of amino acids to reduce the ADCC and/or CDC
activities, preferably 1-20, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12,
1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1 or 2
mutations.
[0067] The present invention has revealed that in some anti-CD40
antibodies, the hinge region of IgG2 is important in expression of
their strong agonistic activities. Replacement of the variable
region or the constant region except the hinge region by a
counterpart of any different subclass, or incorporation of a point
mutation thereinto is expected to not only modulate the ADCC and/or
CDC activities, but increase the productivity of the antibody, its
stability during purification and storage, and its blood
kinetics.
[0068] To produce an antibody drug, the stability of the antibody
during purification and storage is very important. Since the
antibodies so far developed belong mainly to the IgG1 subclass,
conversion of the variable region or the constant region except the
hinge region to a sequence derived from the IgG1 subclass will be
also effective to improve the physical properties of the anti-CD40
agonistic antibodies described above.
[0069] The present invention provides mutants of agonistic
anti-CD40 antibodies and others as follows:
[1] A heavy chain of a monoclonal antibody having an agonistic
activity, which binds to CD40, wherein the heavy chain comprises an
upper hinge and a middle hinge derived from a human IgG2, and a
constant region with at least one amino acid deleted or
substituted, or with at least one amino acid added thereto, said
deletion, substitution or addition being capable of increasing or
decreasing ADCC and/or CDC. [2] The heavy chain according to [1],
wherein the constant region is derived from a human IgG. [3] The
heavy chain according to [2], wherein the human IgG is a human
IgG1. [4] The heavy chain according to [2], wherein the human IgG
is a human IgG2. [5] The heavy chain according to [2], wherein the
human IgG is a human IgG3. [6] The heavy chain according to [2],
wherein the human IgG is a human IgG4. [7] The heavy chain
according to any of [3] to [5], wherein said substitution of amino
acids in the constant region is substitution of proline with serine
at position 331 which is indicated by the EU index as in Kabat et
al. [8] A monoclonal antibody comprising the heavy chain according
to any of [1] to [7]. [9] The heavy chain according to any of [1]
to [7], wherein the heavy chain comprises a variable region from a
heavy chain of a monoclonal antibody produced by the hybridoma
KM341-1-19 (Accession No. FERM BP-7759). [10] A monoclonal antibody
consisting of the heavy chain according to [9] and a light chain
comprising a variable region from a light chain of a monoclonal
antibody produced by the hybridoma KM341-1-19 (Accession No. FERM
BP-7759). [11] The heavy chain according to any of [1] to [7],
wherein the heavy chain comprises a variable region of the
polypeptide represented by SEQ ID NO: 38. [12] A monoclonal
antibody consisting of the heavy chain according to [11] and a
light chain of a monoclonal antibody, wherein the light chain
comprises a variable region of the polypeptide represented by SEQ
ID NO: 40. [13] The heavy chain according to [1], wherein the heavy
chain consists of a remaining portion provided by removing the
signal sequence from the polypeptide represented by SEQ ID NO: 132.
[14] A monoclonal antibody consisting of the heavy chain according
to [13] and a light chain of a monoclonal antibody, wherein the
light chain consists of a remaining portion provided by removing
the signal sequence from the polypeptide represented by SEQ ID NO:
134. [15] The heavy chain according to [1], wherein the heavy chain
is produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 131. [16] The monoclonal
antibody according to [8], wherein the monoclonal antibody is
produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 131 and the polynucleotide
represented by SEQ ID NO: 133. [17] The heavy chain according to
any of [1] to [7], wherein the heavy chain comprises a variable
region from a heavy chain of a monoclonal antibody produced by the
hybridoma 2105 (Accession No. FERM BP-8024). [18] A monoclonal
antibody consisting of the heavy chain according to [17] and a
light chain comprising a variable region from a light chain of a
monoclonal antibody produced by the hybridoma 2105 (Accession No.
FERM BP-8024). [19] The heavy chain according to any of [1] to [7],
wherein the heavy chain comprises a variable region of the
polypeptide represented by SEQ ID NO: 42. [20] A monoclonal
antibody consisting of the heavy chain according to [19] and a
light chain of a monoclonal antibody, wherein the light chain
comprises a variable region of the polypeptide represented by SEQ
ID NO: 44. [21] The heavy chain according to [1], wherein the heavy
chain consists of a remaining portion provided by removing the
signal sequence from the polypeptide represented by SEQ ID NO: 136.
[22] A monoclonal antibody consisting of the heavy chain according
to [21] and a light chain of a monoclonal antibody, wherein the
light chain consists of a remaining portion provided by removing
the signal sequence from the polypeptide represented by SEQ ID NO:
138. [23] The heavy chain according to [1], wherein the heavy chain
is produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 135. [24] The monoclonal
antibody according to [8], wherein the monoclonal antibody is
produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 135 and the polynucleotide
represented by SEQ ID NO: 137. [25] A polynucleotide represented by
SEQ ID NO: 131. [26] A polynucleotide represented by SEQ ID NO:
133. [27] An expression vector having the polynucleotide according
to [25]. [28] An expression vector having the polynucleotide
according to [26]. [29] An expression vector having the
polynucleotides according to [25] and [26]. [30] A host comprising
the expression vector according to [27]. [31] A host comprising the
expression vector according to [28]. [32] A host comprising the
expression vector according to [29]. [33] A process of producing a
heavy chain of a monoclonal antibody, comprising the steps of:
culturing the host according to [30] in a culture medium; and
obtaining a heavy chain of a monoclonal antibody from the culture
and/or the host. [34] A process of producing a monoclonal antibody,
comprising the steps of: culturing the host according to [32] in a
culture medium; and obtaining a monoclonal antibody from the
culture and/or the host. [35] A polynucleotide represented by SEQ
ID NO: 135. [36] A polynucleotide represented by SEQ ID NO: 137.
[37] An expression vector having the polynucleotide according to
[35]. [38] An expression vector having the polynucleotide according
to [36]. [39] An expression vector having the polynucleotides
according to [35] and [36]. [40] A host comprising the expression
vector according to [37]. [41] A host comprising the expression
vector according to [38]. [42] A host comprising the expression
vector according to [39]. [43] A process of producing a heavy chain
of a monoclonal antibody, comprising the steps of: culturing the
host according to [40] in a culture medium; and obtaining a heavy
chain of a monoclonal antibody from the culture and/or the host.
[44] A process of producing a monoclonal antibody, comprising the
steps of: culturing the host according to [42] in a culture medium;
and obtaining a monoclonal antibody from the culture and/or the
host. [45] A process of producing a heavy chain of a monoclonal
antibody having an agonistic activity capable of binding to CD40,
comprising the step of substituting the upper hinge and the middle
hinge of an antibody, which is not either an upper hinge or a
middle hinge derived from a human IgG2, with an upper hinge and a
middle hinge derived from a human IgG2, respectively. [46] A
process of producing a heavy chain of a monoclonal antibody
comprising a variable region, and an upper hinge and a middle hinge
derived from a human IgG2, comprising the step of identifying a
polypeptide forming the variable region, which is from a heavy
chain of a monoclonal antibody capable of binding to CD40. [47] A
process of producing a monoclonal antibody having an agonistic
activity capable of binding to CD40, comprising the step of
substituting the upper hinge and the middle hinge of an antibody,
which is not either an upper hinge or a middle hinge derived from a
human IgG2, with an upper hinge and a middle hinge derived from a
human IgG2, respectively. [48] A process of producing a monoclonal
antibody comprising a variable region, and an upper hinge and a
middle hinge derived from a human IgG2, comprising the step of
identifying a polypeptide forming the variable region, which is
from a heavy chain of a monoclonal antibody capable of binding to
CD40. [49] A pharmaceutical composition comprising the monoclonal
antibody according to [8], [10], [12], [14], [16], [18], [20], [22]
or [24] as an active ingredient. [50] The pharmaceutical
composition according to [49] used for prevention or treatment of a
malignant tumor, a pathogen or an autoimmune disease. [51] A method
of prevention or treatment of a malignant tumor, a pathogen or an
autoimmune disease, comprising administration of the pharmaceutical
composition according to [49] into a mammal. [52] Use of the
monoclonal antibody according to [8], [10], [12], [14], [16], [18],
[20], [22] or [24] for production of a pharmaceutical composition
used for prevention or treatment of a malignant tumor, a pathogen
or an autoimmune disease. [89] A polynucleotide provided by
removing the portion encoding the signal sequence from the
polynucleotide represented by SEQ ID NO: 131. [90] A polynucleotide
provided by removing the portion encoding the signal sequence from
the polynucleotide represented by SEQ ID NO: 133. [91] A
polynucleotide provided by removing the portion encoding the signal
sequence from the polynucleotide represented by SEQ ID NO: 135.
[92] A polynucleotide provided by removing the portion encoding the
signal sequence from the polynucleotide represented by SEQ ID NO:
137.
[0070] The present invention provides an antibody produced by
modification of an agonistic anti-CD40 antibody belonging to the
human IgG2, wherein the modified antibody is a mutant having the
constant region, exclusive of the upper and middle hinges,
substituted with a sequence derived from a different subclass. The
subclass is preferably IgG1. The present invention provides an
antibody produced by modification of an agonistic anti-CD40
antibody belonging to the human IgG2, wherein the modified antibody
is a mutant having the constant region, exclusive of the hinge
region, substituted with a sequence derived from a different
subclass. The subclass is preferably IgG1.
[0071] Herein, reduction in ADCC and CDC activities means reduction
in those activities as compared with the corresponding activities
of an anti-CD40 monoclonal antibody other than the mutants
described above, for example, as compared with the corresponding
activities of a monoclonal antibody produced by the hybridoma
KM341-1-19 (Accession No. FERM BP-7759) or 2105 (Accession No. FERM
BP-8024). The ADCC and CDC activities may be assayed by any known
method, for example, the method described in the Examples herein.
The sequences of variable regions in the heavy and light chains of
a monoclonal antibody will be presented below which is produced by
the hybridoma KM341-1-19 (Accession No. FERM BP-7759) or 2105
(Accession No. FERM BP-8024).
[0072] DNA encoding variable regions in the heavy and light chains
of the KM341-1-19 antibody and the amino acid sequences of the
heavy and light chains will be presented below.
[0073] In the heavy chain nucleotide sequence (SEQ ID NO: 37) of
the KM341-1-19 antibody, the signal sequence is initiated with
adenine (A) at position 50. The boundary between the signal
sequence and the variable region is located between "adenine" ([A])
at position 109 and cytosine (C) at position 110, and the boundary
between the variable region and the constant region is located
between adenine (A) at position 493 and guanine (G) at position 494
(the gene prediction software (Signal P ver.2) was used).
[0074] In the heavy chain amino acid sequence (SEQ ID NO: 38) of
the KM341-1-19 antibody, the boundary between the signal sequence
and the variable region is located between serine (S) at position
20 and glutamine (Q) at position 21, and the boundary between the
variable region and the constant region is located between serine
(S) at position 148 and alanine (A) at position 149.
[0075] Accordingly, the variable region in the heavy chain of the
KM341-1-19 antibody has a nucleotide sequence ranging from cytosine
(C) at position 110 to adenine (A) at position 493, as seen in SEQ
ID NO: 37. Further, the variable region in the heavy chain of the
KM341-1-19 antibody has an amino acid sequence ranging from
glutamine (Q) at position 21 to serine (S) at position 148, as seen
in SEQ ID NO: 38.
[0076] In the light chain nucleotide sequence (SEQ ID NO: 39) of
the KM341-1-19 antibody, the signal sequence is initiated with
adenine (A) at position 29. The boundary between the signal
sequence and the variable region is located between "adenine" ([A])
at position 88 and guanine (G) at position 89, and the boundary
between the variable region and the constant region is located
between adenine (A) at position 400 and "cytosine" ([C]) at
position 401 (the gene prediction software (Signal P ver.2) was
used).
[0077] In the light chain amino acid sequence (SEQ ID NO: 40) of
the KM341-1-19 antibody, the boundary between the signal sequence
and the variable region is located between glycine (G) at position
20 and glutamic acid (E) at position 21, and the boundary between
the variable region and the constant region is located between
lysine (K) at position 124 and "arginine" ([R]) at position
125.
[0078] Accordingly, the variable region in the light chain of the
KM341-1-19 antibody has a nucleotide sequence ranging from guanine
(G) at position 89 to adenine (A) at position 400, as seen in SEQ
ID NO: 39. Further, the variable region in the light chain of the
KM341-1-19 antibody has an amino acid sequence ranging from
glutamic acid (E) at position 21 to lysine (K) at position 124, as
seen in SEQ ID NO: 40.
The heavy chain nucleotide sequence (SEQ ID NO: 37) of the
KM341-1-19 antibody:
TABLE-US-00001 GTCGACGCTGAATTCTGGCTGACCAGGGCAGCCACCAGAGCTCCAG
ACAATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCC
CATGGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGG
ACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCC
GGGGACAGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGC
AGTCCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAG
GTCCAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAATA
ATCATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAGCTGA
ACTCTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGC
ACAGTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTC
TGGGGCCAAGGGACCACGGTCACCGTCTCTTCAGCCTCCACCAAGG
GCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA
GAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAA
CCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGC
ACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAG
CAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTAC
ACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGA
CAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACC
ACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAG
GACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGG
TGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGT
GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAG
CAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGC
ACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAA
CAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG
AGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCAGACG
GCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTG
GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT GAGGATCC
[0079] The heavy chain amino acid sequence (SEQ ID NO: 38) of the
KM341-1-19 antibody:
TABLE-US-00002 MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVIUSQTLSLTCAISG
DSVSSNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRII
INPDTSNNQFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVW
GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP
VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT
CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQ
FNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQP
ENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK
The light chain nucleotide sequence (SEQ ID NO: 39) of the
KM341-1-19 antibody:
TABLE-US-00003 ACTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAG
CTTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAA
TTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGA
AAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTAC
TTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCA
TCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAG
TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTA
GAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACA
CTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTACG
The light chain amino acid sequence (SEQ ID NO: 40) of the
KM341-1-19 antibody:
TABLE-US-00004 MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRAS
QSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT
LTISSLEPEDFAVYYCQQRSNTFGPGTKVDIKRT
[0080] DNA encoding variable regions in the heavy and light chains
of the 2105 antibody and the amino acid sequences of the heavy and
light chains will be presented below.
[0081] In the heavy chain nucleotide sequence (SEQ ID NO: 41) of
the 2105 antibody, the signal sequence is initiated with adenine
(A) at position 70. The boundary between the signal sequence and
the variable region is located between "thymine" ([T]) at position
126 and guanine (G) at position 127, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 495 and guanine (G) at position 496 (the gene
prediction software (Signal P ver.2) was used).
[0082] In the heavy chain amino acid sequence (SEQ ID NO: 42) of
the 2105 antibody, the boundary between the signal sequence and the
variable region is located between cysteine (C) at position 19 and
glutamic acid (E) at position 20, and the boundary between the
variable region and the constant region is located between serine
(S) at position 142 and alanine (A) at position 143.
[0083] Accordingly, the variable region in the heavy chain of the
2105 antibody has a nucleotide sequence ranging from guanine (G) at
position 127 to adenine (A) at position 495, as seen in SEQ ID NO:
41. Further, the variable region in the heavy chain of the 2105
antibody has an amino acid sequence ranging from glutamic acid (E)
at position 20 to serine (S) at position 142, as seen in SEQ ID NO:
42.
[0084] In the light chain nucleotide sequence (SEQ ID NO: 43) of
the 2105 antibody, the signal sequence is initiated with adenine
(A) at position 28. The boundary between the signal sequence and
the variable region is located between "adenine" ([A]) at position
87 and guanine (G) at position 88, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 405 and "cytosine" ([C]) at position 406 (the gene
prediction software (Signal P ver.2) was used).
[0085] In the light chain amino acid sequence (SEQ ID NO: 44) of
the 2105 antibody, the boundary between the signal sequence and the
variable region is located between glycine (G) at position 20 and
glutamic acid (E) at position 21, and the boundary between the
variable region and the constant region is located between lysine
(K) at position 126 and "arginine" ([R]) at position 127.
[0086] Accordingly, the variable region in the light chain of the
2105 antibody has a nucleotide sequence ranging from guanine (G) at
position 88 to adenine (A) at position 405, as seen in SEQ ID NO:
43. Further, the variable region in the light chain of the 2105
antibody has an amino acid sequence ranging from glutamic acid (E)
at position 21 to lysine (K) at position 126, as seen in SEQ ID NO:
44.
The heavy chain nucleotide sequence (SEQ ID NO: 41) of the 2105
antibody:
TABLE-US-00005 CTGAACACAGACCCGTCGACTCCCAGGTGTTTCCATTCAGTGATCA
GCACTGAACACAGAGGACTCACCATGGAGTTGGGACTGAGCTGGAT
TTTCCTTTTGGCTATTTTAAAAGGTGTCCAGTGTGAAGTGCAGCTG
GTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGAC
TCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCA
CTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGT
ATTAGTTGGAATAGTGGTAGCTTGGTGCATGCGGACTCTGTGAAGG
GCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCT
GCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGT
GCAAGAGATAGGCTATTTCGGGGAGTTAGGTACTACGGTATGGACG
TCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAA GG
The heavy chain amino acid sequence (SEQ ID NO: 42) of the 2105
antibody:
TABLE-US-00006 MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGF
TFDDYAMFIWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISR
DNAKNSLYLQMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTT VTVSSASTK
The light chain nucleotide sequence (SEQ ID NO: 43) of the 2105
antibody:
TABLE-US-00007 CTGCTCAGTTAGGACCCAGAGGGAACCATGGAAGCCCCAGCTCAGC
TTCTCTTCCTCCTGCTACTCTGGCTCCCAGATACCACCGGAGAAAT
TGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAA
AGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACT
TAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCAT
CTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGT
GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAG
AGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCCACTG
GCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCAAACGTACGGTG
The light chain amino acid sequence (SEQ ID NO: 44) of the 2105
antibody:
TABLE-US-00008 MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRAS
QSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFT
LTISSLEPEDFAVYYCQQRSHWLTFGGGTKVEIKRTV
[0087] In the heavy chain nucleotide sequence (SEQ ID NO: 131) of
the 341G2Ser, the boundary between the signal sequence and the
variable region is located between "adenine" ([A]) at position 60
and cytosine (C) at position 61, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 444 and guanine (G) at position 445 (the gene
prediction software (Signal P ver.2) was used).
[0088] In the heavy chain amino acid sequence (SEQ ID NO: 132) of
the 341G2Ser, the boundary between the signal sequence and the
variable region is located between serine (S) at position 20 and
glutamine (Q) at position 21, and the boundary between the variable
region and the constant region is located between serine (S) at
position 148 and alanine (A) at position 149.
[0089] Accordingly, the variable region in the heavy chain of the
341G2Ser has a nucleotide sequence ranging from cytosine (C) at
position 61 to adenine (A) at position 444, as seen in SEQ ID NO:
131. Further, the variable region in the heavy chain of the
341G2Ser has an amino acid sequence ranging from glutamine (Q) at
position 21 to serine (S) at position 148, as seen in SEQ ID NO:
132.
The entire heavy chain nucleotide sequence of the 341G2Ser (SEQ ID
NO: 131):
TABLE-US-00009 ATGTCTGTCTCCTTCCTCATCTTCCTGCCCGTGCTGGGCCTCCCAT
GGGGTGTCCTGTCACAGGTCCAACTGCAGCAGTCAGGTCCAGGACT
GGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGG
GACAGTGTCTCTAGCAACAGTGCTACTTGGAACTGGATCAGGCAGT
CCCCATCGAGAGACCTTGAGTGGCTGGGAAGGACATACTACAGGTC
CAAGTGGTATCGTGATTATGTAGGATCTGTGAAAAGTCGAATAATC
ATCAACCCAGACACATCCAACAACCAGTTCTCCCTGCAGCTGAACT
CTGTGACTCCCGAGGACACGGCTATATATTACTGTACAAGAGCACA
GTGGCTGGGAGGGGATTACCCCTACTACTACAGTATGGACGTCTGG
GGCCAAGGGACCACGGTCACCGTCTCCTCAGCTAGCACCAAGGGCC
CATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAG
CACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACA
CCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAG
CGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAG
TTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACC
TGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGAC
ACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGG
ACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGA
CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAG
TTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACC
AGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGGCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAACCAAAGGG
CAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGG
AGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT
CTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCG
GAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCT
CCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCAC
AACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
The entire heavy chain amino acid sequence of the 341G2Ser (SEQ ID
NO: 132):
TABLE-US-00010 MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVIUSQTLSLTCAISGD
SVSSNSATWNWIRQSPSRDLEWLGRTYYRSKWYRDYVGSVKSRIIIN
PDTSNNQFSLQLNSVTPEDTAIYYCTRAQWLGGDYPYYYSMDVWGQG
TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVS
WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDH
KPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRV
VSVLTVVHQDWLNGKEYKCKVSNKGLPASIEKTISKTKGQPREPQVY
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK
[0090] In the light chain nucleotide sequence (SEQ ID NO: 133) of
the 341G2Ser, the boundary between the signal sequence and the
variable region is located between "adenine" ([A]) at position 60
and guanine (G) at position 61, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 372 and "cytosine" ([C]) at position 373 (the gene
prediction software (Signal P ver.2) was used).
[0091] In the light chain amino acid sequence (SEQ ID NO: 134) of
the 341G2Ser, the boundary between the signal sequence and the
variable region is located between glycine (G) at position 20 and
glutamic acid (E) at position 21, and the boundary between the
variable region and the constant region is located between lysine
(K) at position 124 and "arginine" ([R]) at position 125.
Accordingly, the variable region in the light chain of the 341G2Ser
has a nucleotide sequence ranging from guanine (G) at position 61
to adenine (A) at position 372, as seen in SEQ ID NO: 133. Further,
the variable region in the light chain of the 341G2Ser has an amino
acid sequence ranging from glutamic acid (E) at position 21 to
lysine (K) at position 124, as seen in SEQ ID NO: 134.
The entire light chain nucleotide sequence of the 341G2Ser (SEQ ID
NO: 133):
TABLE-US-00011 ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCC
AGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGT
CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAG
AGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGC
TCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCC
CAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC
ATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCA
GCGTAGCAACACTTTCGGCCCTGGGACCAAAGTGGATATCAAACGTA
CGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAG
TTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTA
TCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAAT
CGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGC
ACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGA
GAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCT
CGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
The entire light chain amino acid sequence of the 341G2Ser (SEQ ID
NO: 134):
TABLE-US-00012 MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQ
SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
ISSLEPEDFAVYYCQQRSNTFGPGTKVDIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS
TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0092] In the heavy chain nucleotide sequence (SEQ ID NO: 135) of
the 2105G2Ser, the boundary between the signal sequence and the
variable region is located between "thymine" ([T]) at position 57
and guanine (G) at position 58, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 426 and guanine (G) at position 427 (the gene
prediction software (Signal P ver.2) was used).
[0093] In the heavy chain amino acid sequence (SEQ ID NO: 136) of
the 2105G2Ser, the boundary between the signal sequence and the
variable region is located between cysteine (C) at position 19 and
glutamic acid (E) at position 20, and the boundary between the
variable region and the constant region is located between serine
(S) at position 142 and alanine (A) at position 143.
[0094] Accordingly, the variable region in the heavy chain of the
2105G2Ser has a nucleotide sequence ranging from guanine (G) at
position 58 to adenine (A) at position 426, as seen in SEQ ID NO:
135. Further, the variable region in the heavy chain of the
2105G2Ser has an amino acid sequence ranging from glutamic acid (E)
at position 20 to serine (S) at position 142, as seen in SEQ ID NO:
136.
The entire heavy chain nucleotide sequence of the 2105G2Ser (SEQ ID
NO: 135):
TABLE-US-00013 ATGGAGTTGGGACTGAGCTGGATTTTCCTTTTGGCTATTTTAAAAGG
TGTCCAGTGTGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTAC
AGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACC
TTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGG
CCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCTTGGTGC
ATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCC
AAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACAC
GGCCTTGTATTACTGTGCAAGAGATAGGCTATTTCGGGGAGTTAGGT
ACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCC
TCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTC
CAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGG
ACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTG
ACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACT
CTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCA
CCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAG
GTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTG
CCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAA
AACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGC
GTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTG
GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGG
AGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTT
GTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTC
CAACAAAGGCCTCCCAGCCTCCATCGAGAAAACCATCTCCAAAACCA
AAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGG
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGG
CTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGC
CGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGC
TCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACA
ACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
The entire heavy chain amino acid sequence of the 2105G2Ser (SEQ ID
NO: 136):
TABLE-US-00014 MELGLSWIFLLAILKGVQCEVQLVESGGGLVQPGRSLRLSCAASGFT
FDDYAMHWVRQAPGKGLEWVSGISWNSGSLVHADSVKGRFTISRDNA
KNSLYLQMNSLRAEDTALYYCARDRLFRGVRYYGMDVWGQGTTVTVS
SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL
TSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTK
VDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC
VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTV
VHQDWLNGKEYKCKVSNKGLPASIEKTISKTKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0095] In the light chain nucleotide sequence (SEQ ID NO: 137) of
the 2105G2Ser, the boundary between the signal sequence and the
variable region is located between "adenine" ([A]) at position 60
and guanine (G) at position 61, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 378 and "cytosine" ([C]) at position 379 (the gene
prediction software (Signal P ver.2) was used).
[0096] In the light chain amino acid sequence (SEQ ID NO: 138) of
the 2105G2Ser, the boundary between the signal sequence and the
variable region is located between glycine (G) at position 20 and
glutamic acid (E) at position 21, and the boundary between the
variable region and the constant region is located between lysine
(K) at position 126 and "arginine" ([R]) at position 127.
Accordingly, the variable region in the light chain of the
2105G2Ser has a nucleotide sequence ranging from guanine (G) at
position 61 to adenine (A) at position 378, as seen in SEQ ID NO:
137. Further, the variable region in the light chain of the
2105G2Ser has an amino acid sequence ranging from glutamic acid (E)
at position 21 to lysine (K) at position 126, as seen in SEQ ID NO:
138.
The entire light chain nucleotide sequence of the 2105G2Ser (SEQ ID
NO: 137):
TABLE-US-00015 ATGGAAGCCCCAGCTCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCC
AGATACCACCGGAGAAATTGTGTTGACACAGTCTCCAGCCACCCTGT
CTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAG
AGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGC
TCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCC
CAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACC
ATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCA
GCGTAGCCACTGGCTCACTTTCGGCGGGGGGACCAAGGTGGAGATCA
AACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGAT
GAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAA
CTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCC
TCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG
GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCC
TGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
The entire light chain amino acid sequence of the 2105G2Ser (SEQ ID
NO: 138):
TABLE-US-00016 MEAPAQLLFLLLLWLPDTTGEIVLTQSPATLSLSPGERATLSCRASQ
SVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLT
ISSLEPEDFAVYYCQQRSHWLTFGGGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK
DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
2. Modification of Antagonistic Antibodies
[0097] Anti-CD40 antagonistic antibodies will be more preferable as
therapeutic agent as well as the agonistic antibodies, if they have
not the activities of ADCC and/or CDC, in terms of mechanism of
action. Furthermore, it is important that anti-CD40 antagonistic
antibodies have no activity to induce signals by their in vivo
crosslinking via Fc receptors, even if the ADCC activity cannot be
detected. In other words, it is necessary to confirm that they are
not activate the immunity, and such active antibodies may be
desired as pharmaceutical agent. Anti-CD40 antagonistic antibodies
are promising as therapeutic agent for treating autoimmune diseases
or suppressing rejection in organ transplantation. If they induce
an agonistic activity due to some effect after they are
administered to patients, however weak it may be, the symptoms may
worsen in contrast to the desired therapeutic effect. Thus, an
antibody without any agonistic activity is more preferable as
pharmaceutical agent. In the present invention, incorporation of a
point mutation L235E (means substitution of L at position 235 with
E; similar symbols will be used hereinafter) into IgG4 has been
demonstrated to be effective for in vivo reduction in the agonistic
activity, in the animal test using monkeys. Although IgG4 is a
subclass with low activities of ADCC and CDC, it is reported that
when it was attempted to express IgG4 as recombinant protein in
cells like CHO, its half-molecules were secreted due to a poor S--S
bonding between the heavy chains (Rob C. Aalberse et al.,
Immunology, 105, 9-19, 2002). To overcome this problem,
incorporation of a mutation into the constant region of antibodies
is reported to successfully promote the formation of the S--S
bonding. Therefore, this type of mutation was also evaluated for
its usefulness. Specifically, the mutation of substituting S at
position 228 with P was incorporated (S. Angal et al., Molecular
Immunology, vol. 30, no. 1, 105-108, 1993).
[0098] For antagonistic antibodies as well as agonistic antibodies,
the stability of the antibody during purification and storage is
very important. There may be some methods to create such antibodies
as are physically better while keeping the antagonistic activity.
The antibody pharmaceuticals so far offered commercially belong
mostly to the IgG1 subclass, and they are not reported to be
problematic in pharmaceutical formulation. Based on these facts, it
may be advantageous from the viewpoint of physical properties to
derive the constant region of antibodies from IgG1. In case of
anti-CD40 antibodies, however, they are desirably lower in ADCC and
CDC activities. As a consequence, antibodies having an IgG1-type
constant region modified with some point mutations may be desired.
The mutations described above are useful to create such antibodies.
The IgG1-type constant region may become lower in ADCC and CDC
activities by incorporating point mutation P331G thereinto. It is
also observed that incorporation of point mutation L235E into IgG4
eliminates a slight agonistic activity in vivo to make it
pharmaceutically more active, but makes it physically less stable
at a low pH. Thus, substitution of L235 with an amino acid other
than E may make it physically more functional. As to the 4D11
antibody, it is very similar to the 2B11 antibody with respect to
the structure of its variable region. The 2B11 antibody has a lower
antagonistic activity, but has a higher stability at a low pH,
compared with the 4D11 antibody. If some amino acids derived from
the constant region of 2B11 are incorporated into the 4D11
antibody, based on the above properties, 4D11 may become more
stable. Specifically, point mutation L38V, P58R, G62W, I79M, K81Y,
H87Y, S98A, K109R, V120M or T124A in the heavy chain, or N75S in
the light chain, or a combination thereof may be effective for that
purpose. Specifically, a mutant created by substituting L at
position 38 in the variable region of the heavy chain of the 4D11
antibody with V (abbreviated as L38V; similar symbols will be used
hereinafter), a P58R mutant, a G62W mutant, a I79M mutant, a K81Y
mutant, a H87Y mutant, a S98A mutant, a K109R mutant, a V120M
mutant or a T124A mutant, or a N75S mutant in the light chain, or a
combination thereof may be provided for that purpose.
[0099] The antibody according to the present invention has at least
one mutation of amino acids to reduce the ADCC and/or CDC
activities, preferably 1-15, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7,
1-6, 1-5, 1-4, 1-3, or 1 or 2 mutations.
[0100] The present invention provides mutants of antagonistic
anti-CD40 antibodies and others as follows:
[53] A heavy chain of a monoclonal antibody having an antagonistic
activity capable of binding to CD40, wherein the heavy chain
comprises a constant region with at least one amino acid deleted or
substituted, or with at least one amino acid added thereto, said
deletion, substitution or addition being capable of increasing or
decreasing ADCC and/or CDC. [54] The heavy chain according to [53],
wherein the constant region is derived from a human IgG. [55] The
heavy chain according to [54], wherein the human IgG is a human
IgG1. [56] The heavy chain according to [54], wherein the human IgG
is a human IgG2. [57] The heavy chain according to [54], wherein
the human IgG is a human IgG3. [58] The heavy chain according to
[54], wherein the human IgG is a human IgG4. [59] The heavy chain
according to any of [55], [57] or [58], wherein said substitution
of amino acids in the constant region is substitution of leucine
with glutamic acid at position 235 which is indicated by the EU
index as in Kabat et al. [60] A heavy chain according to any of
[53] to [58], wherein the heavy chain comprises a constant region
with at least one amino acid deleted or substituted, or with at
least one amino acid added thereto, said deletion, substitution or
addition being capable of promoting the formation of the S--S bond
between the heavy chains. [61] The antibody heavy chain according
to [60], wherein said substitution of amino acids in the constant
region is substitution of serine with proline at position 228 which
is indicated by the EU index as in Kabat et al. [62] A monoclonal
antibody comprising the heavy chain according to any of [53] to
[61]. [63] The heavy chain according to any of [53] to [61],
wherein the heavy chain comprises a variable region from a heavy
chain of a monoclonal antibody produced by the hybridoma 4D11
(Accession No. FERM BP-7758). [64] A monoclonal antibody comprising
the heavy chain according to [63] and a light chain comprising a
variable region from a light chain of a monoclonal antibody
produced by the hybridoma 4D11 (Accession No. FERM BP-7758). [65]
The heavy chain according to any of [53] to [61], wherein the heavy
chain comprises a variable region of the polypeptide represented by
SEQ ID NO: 46. [66] A monoclonal antibody consisting of the heavy
chain according to [65] and a light chain of a monoclonal antibody,
wherein the light chain comprises a variable region of the
polypeptide represented by SEQ ID NO: 48. [67] The heavy chain
according to [53], wherein the heavy chain consists of a remaining
portion provided by removing the signal sequence from the
polypeptide represented by SEQ ID NO: 140. [68] A monoclonal
antibody consisting of the heavy chain according to [67] and a
light chain of a monoclonal antibody, wherein the light chain
consists of a remaining portion provided by removing the signal
sequence from the polypeptide represented by SEQ ID NO: 142. [69]
The heavy chain according to [53], wherein the heavy chain is
produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 139. [70] The monoclonal
antibody according to [62], wherein the monoclonal antibody is
produced by a host comprising an expression vector having the
polynucleotide represented by SEQ ID NO: 139 and the polynucleotide
represented by SEQ ID NO: 141. [71] A polynucleotide represented by
SEQ ID NO: 139. [72] A polynucleotide represented by SEQ ID NO:
141. [73] An expression vector having the polynucleotide according
to [71]. [74] An expression vector having the polynucleotide
according to [72]. [75] An expression vector having the
polynucleotides according to [71] and [72]. [76] A host comprising
the expression vector according to [73]. [77] A host comprising the
expression vector according to [74]. [78] A host comprising the
expression vector according to [75]. [79] A process of producing a
heavy chain of a monoclonal antibody, comprising the steps of:
culturing the host according to [76] in a culture medium; and
obtaining a heavy chain of a monoclonal antibody from the culture
and/or the host. [80] A process of producing a monoclonal antibody,
comprising the steps of: culturing the host according to [78] in a
culture medium; and obtaining a monoclonal antibody from the
culture and/or the host. [81] A pharmaceutical composition
comprising the monoclonal antibody according to [62], [64], [66],
[68] or [70] as an active ingredient. [82] The pharmaceutical
composition according to [81] used for prevention or treatment of
transplant rejection, an autoimmune disease, allergy or blood
clotting factor VIII inhibition. [83] A method of prevention or
treatment of transplant rejection, an autoimmune disease, allergy
or blood clotting factor VIII inhibition, which comprises
administering the pharmaceutical composition according to [81] into
a mammal. [84] Use of the monoclonal antibody according to [62],
[64], [66], [68] or [70] for production of a pharmaceutical
composition used for prevention or treatment of transplant
rejection, an autoimmune disease, allergy or blood clotting factor
VIII inhibition. [85] A method of producing a heavy chain of a
monoclonal antibody having an antagonistic activity capable of
binding to CD40, wherein the agonistic activity is lowered,
comprising a step of making deletion or substitution of at least
one amino acid, or addition of at least one amino acid in a
constant region of a heavy chain of a human antibody. [86] The
method according to [85], wherein the constant region is from a
human IgG. [87] The method according to [86], wherein the human IgG
is a human IgG4. [88] The method according to any of [85] to [87],
wherein said substitution of amino acids in the constant region is
substitution of leucine with glutamic acid at position 235 which is
indicated by the EU index as in Kabat et al. [93] A polynucleotide
provided by removing the portion encoding the signal sequence from
the polynucleotide represented by SEQ ID NO: 139. [94] A
polynucleotide provided by removing the portion encoding the signal
sequence from the polynucleotide represented by SEQ ID NO: 141.
[0101] Additionally, the present invention provides the materials
below.
[0102] A mutant of an antagonistic anti-CD40 antibody, comprising
at least one substitution selected from the group consisting of
substitution of L with V at position 38, substitution of P with R
at position 58, substitution of G with W at position 62,
substitution of I with M at position 79, substitution of K with Y
at position 81, substitution of H with Y at position 87,
substitution of S with A at position 98, substitution of K with R
at position 109, substitution of V with M at position 120 and
substitution of T with A at position 124, which substitutions are
all carried out in a variable region of a heavy chain of a
monoclonal antibody produced by the hybridoma 4D11 (Accession No.
FERM BP-7758), and a mutant of an antagonistic anti-CD40 antibody
comprising substitution of N with S at position 75 in a variable
region of a light chain of the 4D11 antibody.
[0103] Herein, reduction in ADCC and CDC activities means reduction
in those activities as compared with the corresponding activities
of an anti-CD40 monoclonal antibody other than the mutants
described above, for example, as compared with the corresponding
activities of a monoclonal antibody produced by the hybridoma 4D11
(Accession No. FERM BP-7758). The ADCC and CDC activities may be
assayed by any known method, for example, the method described in
the Examples herein. The sequences of variable regions in the heavy
and light chains of a monoclonal antibody will be presented below
which is produced by the hybridoma 4D11 (Accession No. FERM
BP-7758).
[0104] DNA encoding variable regions in the heavy and light chains
of the 4D11 antibody and the amino acid sequences of the heavy and
light chains will be presented below, respectively.
[0105] In the heavy chain nucleotide sequence (SEQ ID NO: 45) of
the 4D11 antibody, the boundary between the signal sequence and the
variable region is located between "cytosine" ([C]) at position 93
and cytosine (C) at position 94, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 456 and guanine (G) at position 457 (the gene
prediction software (Signal P ver.2) was used).
[0106] In the heavy chain amino acid sequence (SEQ ID NO: 46) of
the 4D11 antibody, the boundary between the signal sequence and the
variable region is located between serine (S) at position 26 and
glutamine (Q) at position 27, and the boundary between the variable
region and the constant region is located between serine (S) at
position 147 and alanine (A) at position 148.
[0107] Accordingly, the variable region in the heavy chain of the
4D11 antibody has a nucleotide sequence ranging from cytosine (C)
at position 94 to adenine (A) at position 456, as seen in SEQ ID
NO: 45. Further, the variable region in the heavy chain of the 4D11
antibody has an amino acid sequence ranging from glutamine (Q) at
position 27 to serine (S) at position 147, as seen in SEQ ID NO:
46.
[0108] In the light chain nucleotide sequence (SEQ ID NO: 47) of
the 4D11 antibody, the boundary between the signal sequence and the
variable region is located between "thymine" ([T]) at position 124
and guanine (G) at position 125, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 442 and "cytosine" ([C]) at position 443 (the gene
prediction software (Signal P ver.2) was used).
[0109] In the light chain amino acid sequence (SEQ ID NO: 48) of
the 4D11 antibody, the boundary between the signal sequence and the
variable region is located between cytosine (C) at position 22 and
alanine (A) at position 23, and the boundary between the variable
region and the constant region is located between lysine (K) at
position 128 and "arginine" ([R]) at position 129.
[0110] Accordingly, the variable region in the light chain of the
4D11 antibody has a nucleotide sequence ranging from guanine (G) at
position 125 to adenine (A) at position 442, as seen in SEQ ID NO:
47. Further, the variable region in the light chain of the 4D11
antibody has an amino acid sequence ranging from alanine (A) at
position 23 to lysine (K) at position 128, as seen in SEQ ID NO:
48.
The heavy chain nucleotide sequence (SEQ ID NO: 45) of the 4D11
antibody:
TABLE-US-00017 ATATGTCGACGAGTCATGGATCTCATGTGCAAGAAAATGAAGCACCT
GTGGTTCTTCCTCCTGCTGGTGGCGGCTCCCAGATGGGTCCTGTCCC
AGCTGCAGCTGCAGGAGTCGGGCCCAGGACTACTGAAGCCTTCGGAG
ACCCTGTCCCTCACCTGCACTGTCTCTGGCGGCTCCATCAGCAGTCC
TGGTTACTACGGGGGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGG
AGTGGATTGGGAGTATCTATAAAAGTGGGAGCACCTACCACAACCCG
TCCCTCAAGAGTCGAGTCACCATATCCGTAGACACGTCCAAGAACCA
GTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCAGACACGGCTGTGT
ATTACTGTACGAGACCTGTAGTACGATATTTTGGGTGGTTCGACCCC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGC
The heavy chain amino acid sequence (SEQ ID NO: 46) of the 4D11
antibody:
TABLE-US-00018 MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLT
CTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSR
VTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGT LVTVSSAS
The light chain nucleotide sequence (SEQ ID NO: 47) of the 4D11
antibody:
TABLE-US-00019 AGATCTTAAGCAAGTGTAACAACTCAGAGTACGCGGGGAGACCCACT
CAGGACACAGCATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTT
CTGCTGCTCTGGCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCA
GTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCA
CTTGCCGGGCAAGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAG
CAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAA
TTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGA
CAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCA
ACTTATTACTGTCAACAGTTTAATAGTTACCCGACGTTCGGCCAAGG
GACCAAGGTGGAAATCAAACGTACG
The light chain amino acid sequence (SEQ ID NO: 48) of the 4D11
antibody:
TABLE-US-00020 MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRA
SQGISSALAWYQQKPGKAPKWYDASNLESGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRT
[0111] In the heavy chain nucleotide sequence (SEQ ID NO: 139) of
the 4D11 antibody G4PE, the boundary between the signal sequence
and the variable region is located between "cytosine" ([C]) at
position 78 and cytosine (C) at position 79, and the boundary
between the variable region and the constant region is located
between adenine (A) at position 441 and guanine (G) at position 442
(the gene prediction software (Signal P ver.2) was used).
[0112] In the heavy chain amino acid sequence (SEQ ID NO: 140) of
the 4D11 antibody, the boundary between the signal sequence and the
variable region is located between serine (S) at position 26 and
glutamine (Q) at position 27, and the boundary between the variable
region and the constant region is located between serine (S) at
position 147 and alanine (A) at position 148.
[0113] Accordingly, the variable region in the heavy chain of the
4D11 antibody has a nucleotide sequence ranging from cytosine (C)
at position 79 to adenine (A) at position 441, as seen in SEQ ID
NO: 139. Further, the variable region in the heavy chain of the
4D11 antibody has an amino acid sequence ranging from glutamine (Q)
at position 27 to serine (S) at position 147, as seen in SEQ ID NO:
140.
The entire heavy chain nucleotide sequence (SEQ ID NO: 139) of the
4D11G4PE:
TABLE-US-00021 ATGGATCTCATGTGCAAGAAAATGAAGCACCTGTGGTTCTTCCTCCT
GCTGGTGGCGGCTCCCAGATGGGTCCTGTCCCAGCTGCAGCTGCAGG
AGTCGGGCCCAGGACTACTGAAGCCTTCGGAGACCCTGTCCCTCACC
TGCACTGTCTCTGGCGGCTCCATCAGCAGTCCTGGTTACTACGGGGG
CTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGAGTA
TCTATAAAAGTGGGAGCACCTACCACAACCCGTCCCTCAAGAGTCGA
GTCACCATATCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCT
GAGCTCTGTGACCGCCGCAGACACGGCTGTGTATTACTGTACGAGAC
CTGTAGTACGATATTTTGGGTGGTTCGACCCCTGGGGCCAGGGAACC
CTGGTCACCGTCTCCTCAGCTAGCACCAAGGGGCCATCCGTCTTCCC
CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGG
AACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCT
ACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCT
CCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAG
CCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCC
CCCATGCCCACCATGCCCAGCACCTGAGTTCGAGGGGGGACCATCAG
TCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGG
ACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCC
CGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTG
GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGA
AAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTAC
ACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGT
GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCC
GTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGT
GGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGA
TGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCTCTGGGTAAATGA
The entire heavy chain amino acid sequence (SEQ ID NO: 140) of the
4D11G4PE:
TABLE-US-00022 MDLMCKKMKHLWFFLLLVAAPRWVLSQLQLQESGPGLLKPSETLSLT
CTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYHNPSLKSR
VTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGT
LVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSW
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHK
PSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV
VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK
[0114] In the light chain nucleotide sequence (SEQ ID NO: 141) of
the 4D11G4PE, the boundary between the signal sequence and the
variable region is located between "thymine" ([T]) at position 66
and guanine (G) at position 67, and the boundary between the
variable region and the constant region is located between adenine
(A) at position 384 and "cytosine" ([C]) at position 385 (the gene
prediction software (Signal P ver.2) was used).
[0115] In the light chain amino acid sequence (SEQ ID NO: 142) of
the 4D11G4PE, the boundary between the signal sequence and the
variable region is located between cytosine (C) at position 22 and
alanine (A) at position 23, and the boundary between the variable
region and the constant region is located between lysine (K) at
position 128 and "arginine" ([R]) at position 129.
[0116] Accordingly, the variable region in the light chain of the
4D11 G4PE has a nucleotide sequence ranging from guanine (G) at
position 67 to adenine (A) at position 384, as seen in SEQ ID NO:
141. Further, the variable region in the light chain of the 4D11
antibody has an amino acid sequence ranging from alanine (A) at
position 23 to lysine (K) at position 128, as seen in SEQ ID NO:
142.
The entire light chain nucleotide sequence (SEQ ID NO: 141) of the
4D11G4PE:
TABLE-US-00023 ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGGCTTCTGCTGCTCTG
GCTCCCAGGTGCCAGATGTGCCATCCAGTTGACCCAGTCTCCATCCT
CCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCA
AGTCAGGGCATTAGCAGTGCTTTAGCCTGGTATCAGCAGAAACCAGG
GAAAGCTCCTAAGCTCCTGATCTATGATGCCTCCAATTTGGAAAGTG
GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACT
CTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTATTACTG
TCAACAGTTTAATAGTTACCCGACGTTCGGCCAAGGGACCAAGGTGG
AAATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCT
GAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA
ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGGTGAGCAA
AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC
AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TGA
The entire light chain amino acid sequence (SEQ ID NO: 142) of the
4D11G4PE:
TABLE-US-00024 MDMRVPAQLLGLLLLWLPGARCAIQLTQSPSSLSASVGDRVTITCRA
SQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSRFSGSGSGTDFT
LTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRTVAAPSVFIFPP
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
3. Definition
[0117] The terms used herein will be defined below.
[0118] "CD40" described in the present invention refers to a
polypeptide having the amino acid sequence described in E. A. Clark
et al., Proc. Natl. Acad. Sci. USA 83: 4494, 1986, or I.
Stamenkovic et al., EMBO J. 8: 1403, 1989, and particularly, an
antigenic polypeptide expressed on the surface of B cells, DC,
macrophages, endothelial cells, epithelial cells or tumor cells
derived from them.
[0119] "An anti-CD40 antibody" refers to any monoclonal antibody to
a cell-expressed CD40, a full-length CD40 or a partial-length
CD40.
[0120] In addition, "an antibody" of the invention is derived from
genes (collectively called antibody genes) encoding a heavy chain
variable region and a heavy chain constant region, as well as a
light chain variable region and a light chain constant region which
together constitute an immunoglobulin. Human immunoglobulins are
grouped into 5 different classes consisting of IgG, IgA, IgM, IgD,
and IgE. Further, IgG is composed of 4 different subclasses, IgG1,
IgG2, IgG3 and IgG4, while IgA is composed of 2 different
subclasses, IgA1 and IgA2. IgG1, IgG2, IgG3 and IgG4 are located in
14q32, 33 of the human chromosomes. The fundamental structure of
immunoglobulin is composed of two homologous L chains (light
chains) and two homologous H chains (heavy chains). The class and
subclass of an immunoglobulin is determined by its H chains. The
antibody according to the present invention may comprise any class,
any subclass or any isotype of immunoglobulin. "A functional
fragment" of the inventive antibody refers to a portion (partial
fragment) of the antibody defined above that is active singly or
multiply on an antigen to the antibody, including, for example,
F(ab').sub.2, Fab', Fab, Fv, disulfide-stabilized FV, single-chain
FV (scFV) and a multimer thereof (D. J. King, Applications and
Engineering of Monoclonal Antibodies, 1998, T. J. International
Ltd.).
[0121] Up to now, it has been known that IgG1 includes J00228,
Z17370 and Y14737, IgG2 includes J00230, AJ250170, AF449616,
AF449617, AF449618, Z49802 and Z49801, IgG3 includes M12958,
K01313, X16110, X99549, AJ390236, AJ390237, AJ390238, AJ390241,
AJ390242, AJ390246, AJ390247, AJ390252, AJ390244, AJ390254,
AJ390260, AJ390262, AJ390272, AJ390276 and AJ390279, and IgG4
includes K01316, AJ001563 and AJ001564 (the symbols listed above
indicates accession numbers of the genes).
[0122] In the present invention, CH1, hinge, CH2 and CH3 each
denote a portion of the heavy-chain constant region of any
antibody, and are based on the EU index as in Kabat et al. (Kabat
et al., Sequences of proteins of immunological interest, 1991 Fifth
edition). By definition, CH1 ranges from 118 to 215 by the EU
index, hinge ranges from 216 to 230 by the EU index, CH2 ranges
from 231 to 340 by the EU index, and CH3 ranges from 341 to 446 by
the EU index.
[0123] The "human antibody" of the present invention means an
antibody which is an expression product of a human-derived antibody
gene.
[0124] "Agonistic" refers to an action of enhancing binding of a
ligand to CD40 expressed on the surface of such cells as B cells,
tumor cells or dendritic cells, or an action of providing the
CD40-expressing cells with at least one effect which the CD40
ligand makes on the CD40-expressing cells. "An agonistic antibody"
refers to an antibody having such an agonistic action. An example
of the effects provided for the CD40-expressing cells is to promote
the expression of CD95.
[0125] "Antagonistic" refers to an action of inhibiting binding of
the ligand to CD40 expressed on the surface of such cells as B
cells, tumor cells or dendritic cells, or an action of neutralizing
at least one effect which the CD40 ligand makes on the
CD40-expressing cells. "An antagonistic antibody" refers to an
antibody having such an antagonistic action. An example of the
effects provided for the CD40-expressing cells is to suppress the
proliferation of B cells or the production of antibodies.
[0126] The present application clearly presents antibodies, or
heavy chain or light chain variable regions thereof by amino acid
sequences. The present invention also encompasses the amino acid
sequences with at least one amino acid deleted or substituted, or
added thereto, preferably 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3,
or 1 or 2 amino acids.
[0127] The present application clearly presents genes which encode
antibodies, or heavy chain or light chain variable regions thereof
by nucleotide sequences. The present invention also encompasses the
nucleotide sequences with at least one nucleotide deleted or
substituted, or added thereto, preferably 1-10, 1-9, 1-8, 1-7, 1-6,
1-5, 1-4, 1-3, or 1 or 2 amino acids.
[0128] The anti-CD40 antibody according to the present invention
can be provided by incorporating an antibody gene into an
expression vector, transfecting the vector into a suitable host
cell, harvesting the antibody from the cultured cells or the
supernatant, and purifying it.
[0129] The vector may be a phage or a plasmid which can replicate
in the host cell by itself or can be integrated into the chromosome
of the host cell. The plasmid DNA may be derived from Escherichia
coli, Bacillus subtilis or a yeast, while the phage DNA may be from
2, phage.
[0130] The host cell for transformation is not particularly limited
if it can express the target gene. Examples of the host cell may
include bacteria (Escherichia coli, Bacillus subtilis, etc.),
yeasts, animal cells (COS cell, CHO cell, etc.) and insect
cells.
[0131] There are known modes of transferring the gene into the host
cells, including any mode, such as mediation by calcium ion,
electroporation, spheroplast fusion, mediation by lithium acetate,
calcium phosphate transfection or lipofection. In order to transfer
the gene into an animal, as described later, the modes include
microinjection; electroporation or lipofection for ES cells; and
nuclear transplantation.
[0132] In the present invention, "culture" refers to (a) culture
supernatant, (b) cultured cells, cultured biomass or disrupted
matter thereof, or (c) secretion of transformant To culture the
transformant, a medium suitable for the host is used and static
culture, roller bottle culture or something else may be
employed.
[0133] After the culture, if the desired antibody protein is
produced within the biomass or cells, the antibody is harvested by
disrupting the biomass or cells. If the desired antibody is
produced out of the biomass or cells, the culture solution is used
as it is or after it is separated from the biomass or cells by
centrifugation or other means. Thereafter, a biochemical process
utilizing any chromatography, which is appropriate for
separation/purification of proteins, is employed alone or
optionally in combination with another to separate/isolate the
desired antibody from the culture.
[0134] Furthermore, the technology of creating a transgenic animal
may be used to produce a transgenic animal that is a host animal
having the gene integrated into an endogenous gene, such as a
transgenic bovine, a transgenic goat, a transgenic sheep or a
transgenic pig (Wright, G., et al., (1991) Bio/Technology 9,
830-834) and a large amount of a monoclonal antibody derived from
the antibody gene can be obtained from the milk secreted from the
transgenic animal. The culture of a hybridoma in vitro can be done
by using a known nutrient medium or any nutrient medium
derivatively prepared from known basic media as used to grow,
maintain and store the hybridoma and to produce a monoclonal
antibody in the supernatant, depending on the properties of the
cultured hybridoma, the purpose of the study and the culture
method.
4. Antibody Properties
(1) Agonistic Antibodies
[0135] The mutant of the agonistic antibody according to the
present invention may activate the immune system without injuring
immunocompetent cells, since it has an ADCC and/or CDC activity
equal to or lower than the original antibody, while keeping an
agonistic activity. It is thus expected that the mutant exhibits
the immunoactivating action which is equal to or higher than the
original antibody and the cytotoxicity to CD40-expressing cells
which is equal to or lower than the original antibody.
(2) Antagonistic Antibodies
[0136] The mutant of the antagonistic anti-CD40 antibody according
to the present invention has the reduced ADCC and/or CDC activity
compared to the unmodified antibody, while keeping a suppressive
activity against immunoactivating signals induced by CD40L. It is
also expected to decrease the activity of signal induction in vivo
which is considered to occur via Fc receptors.
5. Pharmaceutical Compositions
[0137] A pharmaceutical composition containing a formulation of the
purified antibody according to the present invention is also within
the scope of the present invention. The pharmaceutical composition
may preferably contain a physiologically acceptable diluent or
carrier in addition to the antibody, and may be a mixture thereof
with a different antibody or a different drug such as an antibiotic
agent. The suitable carrier may include, but not limited to,
physiological saline, phosphate buffered physiological saline,
phosphate buffered physiological saline glucose solution and
buffered physiological saline. Alternatively, the antibody may be
freeze-dried for storage and when it is used, reconstituted in an
aqueous buffer as described above. The pharmaceutical composition
may be administered via the oral route, or the parenteral route,
such as intravenous, intramuscular, subcutaneous or intraperitoneal
injection or dosing.
[0138] A single effective dose, which is a combination of the
antibody of the present antibody with a suitable diluent and a
physiologically acceptable carrier, is from 0.0001 mg to 100 mg per
kg of body weight, and it may be taken at a time interval of from 2
days to 8 weeks.
[0139] When the pharmaceutical composition of the present antibody
is an agonistic antibody, it is used as immunostimulant (antiviral
or anti-infective agent) for pathogens including, for example,
hepatitis A, B, C, D or E virus, HIV, influenza virus, simple
herpes virus, cytomegalovirus, EB virus, papiloma virus, chlamydia,
mycoplasma, toxoplasma, malaria, trypanosome and tubercle bacillus;
antitumor agent for malignant tumors having cancer cells with CD40
expressed, including, for example, pancreatic cancer, bladder
cancer, lymphoma (e.g., Hodgkin's lymphoma), leukemia, malignant
melanoma, pancreatic cancer, lung cancer, ovarian cancer, bladder
cancer, breast cancer, colon cancer, prostatic cancer, and head and
neck cancer; and therapeutic agent for autoimmune diseases such as
rheumatism. The pharmaceutical composition may be used for a
combination of the above diseases. It may be also used in
combination as adjuvant for a cancer-specific peptide. When the
pharmaceutical composition is an antagonistic antibody, on the
other hand, it is useful as: immunosuppressant in organ
transplantation (preventive or therapeutic agent for rejection in
transplantation of pancreatic islets, kidney or something else, or
GVHD), therapeutic agent for autoimmune diseases (e.g., rheumatism,
psoriasis, chronic ulcerative colitis, Crohn's disease, systemic
lupus erythematosus, multiple sclerosis, myasthenia, scleroderma,
antiphospholipid antibodies syndrome, autoimmune hepatitis,
idiopathic thrombocytopenic purpura, Behcet's syndrome,
arteriosclerosis, nephritis and respiratory distress syndrome),
therapeutic agent for allergy (e.g., asthma), and therapeutic agent
for blood clotting factor VIII inhibition. The pharmaceutical
composition may be used for a combination of the above
diseases.
6. Epitopes
[0140] The binding epitopes of CD40 were determined for the
KM341-1-19 and 2105 antibodies having a superior agonistic
activity, and for the 4D11 antibody having a superior antagonistic
activity, respectively (Example 2). The present invention provides
antibodies having an agonistic or antagonistic activity which have
a different variable region sequence from those of the above
antibodies but recognize the same epitope as one of the above
antibodies. These antibodies can be obtained in such a procedure as
described below.
[0141] When it is intended to acquire an agonistic anti-CD40
antibody recognizing the same epitope as the KM341-1-19 antibody,
for example, mice or the likes are immunized with CD40 to provide
monoclonal antibodies, from which some monoclonal antibodies
competing with the KM341-1-19 antibody to bind to CD40 are screened
according to the standard procedure. From the screened antibodies,
an antibody having the same pattern of binding to the peptide as
the KM341-1-19 antibody is selected according to the method
described in Example 2.
[0142] The present invention will be described in more detail below
with reference to examples. However, the present invention is not
limited to embodiments described in the examples.
Example 1
Expression and Purification of Antibody and Antigen Proteins
[0143] A vector plasmid containing a variable region of an antibody
was transfected into CHO cells (ATCC), and antibody-expressing
cells were selected by G418 to prepare a stable expression cell
line.
[0144] A mutant antigen was expressed by transiently introducing a
vector into HEK cells (ATCC).
[0145] An anti-CD40 antibody was purified from the above culture
supernatant by the following method. The culture supernatant
containing an anti-CD40 antibody was affinity purified in a Hyper
D.RTM. Protein A column (manufactured by NGK Insulators, Ltd.) or
in case of mouse IgG1 purification, a Protein G column (Amersham
Pharmacia Biotech) according to the attached instruction using
PBS(-) as an adsorption buffer and a 0.1 M sodium citrate buffer
(pH 3) as an elution buffer. The eluted fraction was adjusted to
about pH 7.2 by addition of a 1 M Tris-HCl (pH 8.0) or Na2HPO4
solution. The prepared antibody solution was substituted with
PBS(-) using a dialysis membrane (10,000 cuts, manufactured by
Spectrum Laboratories, Inc.) or an SP column (Amersham Pharmacia
Biotech), and filtered and sterilized using a membrane filter
MILLEX.RTM.-GV with a pore diameter of 0.22 .mu.m (manufactured by
Millipore Corp.). The concentration of the purified antibody was
calculated by measurement of the absorbance at 280 nm, taking 1
mg/ml as 1.450D.
Example 2
Determination of Epitopes
[0146] A 13-mer peptide covering amino acid 175 (SEQ ID NO: 1) in
an extracellular region of CD40 was shifted by two amino acids each
to synthesize 82 peptides in total (SEQ ID NOS: 49 to 130) as spots
from the C-terminal on a cellulose membrane and acetylate the
N-terminal thereof (Jerini AG, Germany). The reaction thereafter
was carried out based on a conventional Western analysis (see
Reineke, U. et al. (2001), "Epitope mapping with synthetic peptides
prepared by SPOT synthesis." Antibody Engineering (Springer Lab
Manual) Eds.: Kontermann/Dubel, 433-459, for example). In the
analysis, coloring intensity of each spot was quantified using
LumiImager.TM. (Boehringer-Mannheim Corp.) (FIGS. 1A-1, A-2, B-1
and B-2).
[0147] The results confirmed that an 4D11 antibody strongly
recognizes the 20th to 24th and 41st peptides, a 2105 antibody
strongly recognizes the 12th to 23rd and 64th peptides, a
KM341-1-19 antibody strongly recognizes the 41st and 42nd peptides,
KM643-4-11 strongly recognizes the 43rd peptide, F72 strongly
recognizes the 75th peptide, 110 strongly recognizes the 64th
peptide, F4-465 strongly recognizes the 34th, 35th, 54th, 55th,
65th, 66th and 75 peptides, KM281-1-10 strongly recognizes the
21st, 24th, 64th and 75th peptides, 2B11 (novel antibody) strongly
recognizes the 21st, 24th and 64th peptides, and F76 (novel
antibody) strongly recognizes the 21st, 35th, 51st and 52nd
peptides.
[0148] In order to confirm the binding site of the anti-CD40
antibody, a CD40-FC fusion protein having a mutation introduced
thereinto was prepared, and the binding ability thereto was
examined by ELISA. Since the anti-CD40 antibody does not
cross-react with mouse B cells, five CD40Fc fusion proteins were
prepared by partially converting the amino acid sequence into that
of mouse CD40. Binding of the antibody to the antigens was
examined. The method for preparing the mutant CD40-FC fusion
proteins is shown below. The mutation site was prepared by
introducing a mouse CD40 sequence into a part to which the antibody
strongly binds of the peptide sequence. CD40mut1 converted EFTE at
a site corresponding to the 15th peptide into ALEK, CD40mut2
converted LDT at a site corresponding to the 21st peptide into SAQ,
CD40mut3 converted TH at a site corresponding to the 24th peptide
into IR, CD40mut4 converted EEGW at a site corresponding to the
42nd peptide into KEGQ, and CD40mut5 converted VSSA at a site
corresponding to the 64th peptide into QSSL. The mutants were
prepared according to a gene engineering technique (FIGS. 2A, B and
C). The analysis results confirmed that the 2105 antibody has
extremely reduced binding ability to CD40mut1. The results also
confirmed that the 4D11 antibody and 2B11 have reduced binding
ability to CD40mut2.
Example 3
Binding Activity of Anti-CD40 Agonistic Antibody to Ramos Cells
[0149] A Ramos cell line was suspended in a PBS staining buffer
(SB) containing 0.1% NaN.sub.3 and 2% FCS at a concentration of
2.times.10.sup.6/ml. The cell suspension (100 .mu.l/well) was
dispensed to a 96-well round-bottom plate (manufactured by Becton,
Dickinson and Company). Each hybridoma culture supernatant (50
.mu.l) was added, and incubated at an ice temperature for 30
minutes. A human IgG1 antibody to human serum albumin as a negative
control was adjusted to a concentration of 2 .mu.g/ml in a
hybridoma culture medium, added in an amount of 50 .mu.l, and then
incubated at an ice temperature for 15 minutes. After washing the
plate with SB, 50 .mu.l of a 250-fold diluted R-PE fluorescently
labeled anti-human antibody (manufactured by Southern Biotechnology
Associates, Inc.) was added, and incubated at an ice temperature
for 15 minutes. After washing the plate with SB twice, it was
suspended in 300 to 500 .mu.l of a FACS buffer, and fluorescence
intensity of each cell was measured using FACS (FACSort, FACScan,
manufactured by Becton, Dickinson and Company).
Example 4
Evaluation of Agonistic Activity of Anti-CD40 Agonistic Antibody to
Ramos Cells
[0150] 5.0.times.10.sup.5 cells/ml of a Ramos cell suspension was
seeded on a 96-well plate at 100 .mu.l/well. A hybridoma culture
supernatant or purified antibody was diluted to 20 .mu.g/ml in a
medium, and the dilution was added to the 96-well plate at a
concentration of 100 .mu.l/well. After culturing overnight, cells
were harvested, and R-PE labeled anti-CD95 antibody (Pharmingen NJ)
was used for the cells. Analysis was carried out using FACScan or
FACSsort (Becton, Dickinson and Company).
Example 5
Inhibition of CD95 Expression by Anti-CD40 Antagonistic Antibody in
Ramos Cells
[0151] 1.0.times.10.sup.6 cells/ml of a Ramos cell suspension was
seeded on a 96-well plate at 50 .mu.l/well. A hybridoma culture
supernatant or purified antibody was adjusted to 2 .mu.g/ml in a
medium, and the medium was added to the 96-well plate at 100
.mu.l/well. 4 .mu.g/ml of a soluble CD40 ligand (Alexis
Corporation) and 4 .mu.g/ml of an anti-FLAG antibody (M2, Sigma)
were added to a medium, and the medium was added to a 96-well plate
at 50 .mu.l/well. After culturing overnight, cells were harvested,
and an R-PE labeled anti-CD95 antibody (Pharmingen NJ) was used for
the cells. Analysis was carried out using FACS.
Example 6
Measurement of CDC Activity in Anti-CD40 Antibody
[0152] In the CDC assay, 2,000 Cr.sup.51-labeled target cells and a
human serum-derived complement (manufactured by Sigma Co.) or
rabbit serum-derived complement (Cedarlane Laboratories Limited,
Ontario, Canada) at a final concentration of 5% were cultured in a
round-bottom 96-well plate in a total volume of 200 .mu.L together
with the antibody at various concentrations at 37.degree. C. in the
presence of 5% CO.sub.2 for two hours.
[0153] After culturing, the plate was centrifuged to cause the
cells to precipitate, and then 50 .mu.L of the supernatant was
transferred to a 96-well plate including a powder scintillator
(Lumaplate.TM.-96: manufactured by Packard Instrument Co., Inc.)
and dried at 55.degree. C. for 1.5 hours. After confirming that the
plate was dried, it was covered with a special cover
(TopSeal.TM.-A: 96-well microplates: manufactured by Packard
Instrument Co., Inc.), and the g-ray dose was measured with a
scintillation counter (TopCount: manufactured by Packard Instrument
Co., Inc.).
Example 7
Measurement of ADCC Activity of Anti-CD40 Antibody
[0154] As antibody-mediated cytotoxicity, cytotoxicity to target
cells in the presence of cells having killer activity such as NK
cells or neutrophils and an antibody (Antibody-Dependent Cellular
Cytotoxicity, hereinafter ADCC), and cytotoxicity to target cells
in the presence of a complement and an antibody
(Complement-Dependent Cytotoxicity, hereinafter CDC) were measured.
hIgG was used as a control.
[0155] The measurement method is simply described as follows.
Radioactive chromium (Cr.sup.51) was incorporated into the
cytoplasm of the target cells, and the amount of Cr.sup.51 released
in the culture solution by cell death was measured as a .gamma.-ray
dose.
[0156] Specifically, 10.sup.5 target cells of a Burkitt's lymphoma
cell line Raji (ATCC CCL-86) were suspended in 15 .mu.L of fetal
calf serum (FCS). 50 .mu.L (37 MBq/mL) of Cr.sup.51-labeled sodium
chromate (manufactured by PerkinElmer, Inc.: hereinafter referred
to as Cr.sup.51) was added to the suspension, and the cells were
cultured at 37.degree. C. for one hour. Next, 10 mL of a medium was
added, and the medium was discarded by centrifugation. This
operation was repeated three times to remove Cr.sup.51 not
incorporated in the cells.
[0157] In the ADCC assay, 2,000 Cr.sup.51-labeled target cells and
200,000 healthy human peripheral blood mononuclear leukocytes
obtained by the method described in Example 6 were cultured in a
round-bottom 96-well plate (manufactured by Falcon) in a total
volume of 200 .mu.L together with the antibody at various
concentrations at 37.degree. C. in the presence of 5% CO.sub.2 for
four hours.
[0158] After culturing, the plate was centrifuged to cause the
cells to precipitate, and then 50 IAL of the supernatant was
transferred to a 96-well plate including a powder scintillator
(Lumaplate.TM.-96: manufactured by Packard Instrument Co., Inc.)
and dried at 55.degree. C. for 1.5 hours. After confirming that the
plate was dried, the plate was covered with a special cover
(TopSeal.TM.-A: 96-well microplates: manufactured by Packard
Instrument Co., Inc.), and the g-ray dose was measured with a
scintillation counter (TopCount: manufactured by Packard Instrument
Co., Inc.).
Example 8
Preparation and Activity Evaluation of Anti-CD40 Agonistic Antibody
P331S Mutant
[0159] Gene cloning of anti-CD40 agonistic antibodies KM341-1-19
and 2105 is described in WO 02/099186. It is reported that CDC
activity is reduced by converting Pro at position 331 in the IgG2
constant region into Ser. To reduce CDC activity of the KM341-1-19
antibody and the 2105 antibody, a P331S mutation was introduced
into the IgG2 constant region thereof.
[0160] The human IgG1 constant region of an antibody-expressing
vector N5KG1-Val Lark (IDEC Pharmaceuticals: hereinafter
abbreviated to N5KG1) was substituted with human IgG2 to prepare
N5KG2, and the Pro at position 331 of IgG2 was converted into Ser
to prepare a mutation. cDNA cloning of the IgG2 constant region was
carried out by harvesting KM341-1-19 hybridoma by centrifugation,
adding TRIZOL (Gibco BRL), and extracting total RNA according to
the instruction. The antibody cDNA variable region was cloned using
a SMART RACE cDNA amplification kit of Clontech Laboratories, Inc.
according to the attached instruction. 1st strand cDNA was prepared
using 5 .mu.g of total RNA as a template. PCR was carried out with
tnIgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGC (SEQ ID NO: 2)G
and tnIgG2Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ ID: 3)
as primer sequences using a ZtaqPCR kit (Takara) in 30 cycles each
consisting of reaction at 98.degree. C. for 1 second, at 55.degree.
C. for 30 seconds and at 72.degree. C. for 1 minute to amplify the
gene. After the reaction, the amplified product was purified by a
QIAGEN PCR purification kit, digested with NheI and BamHI, and
incorporated into N5KG1 to confirm the sequence. This vector was
defined as N5KG2.
[0161] N5KG2Ser (with Pro at position 331 converted into Ser) was
prepared as follows. Reaction at 98.degree. C. for 1 second, at
60.degree. C. for 30 seconds and at 72.degree. C. for 30 seconds
was carried out 15 times using N5KG2 as a template and primers
IgG3Nhe: atatGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCG (SEQ ID NO: 4)
and G2Ser2: GTTTTCTCGATGGAGGCTGGGAGGCC (SEQ ID NO: 5). At the same
time, reaction at 98.degree. C. for 1 second, at 60.degree. C. for
30 seconds and at 72.degree. C. for 30 seconds was carried out 15
times using N5KG2 as a template and primers IgG2Bam:
atatggatccTCATTTACCCGGAGACAGGGAGAGGCTC (SEQ ID NO: 6) and G2Ser1:
GGCCTCCCAGCCTCCATCGAGAAAAC (SEQ ID NO: 7). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers IgG3Nhe and IgG2Bam were added to the mixture, and
the same reaction was carried out 15 times. The amplified DNA
fragment was cleaved with NheI and BamHI, and substituted with the
IgG1 constant region of the N5KG1 vector (N5KG2Ser). The fragment
containing the sequence of the antibody variable region digested
with BglII and NheI was incorporated into the N5KG2Ser vector.
[0162] The antibody expressed and purified by the above method was
evaluated in terms of binding ability to Ramos cells (FIG. 3A) and
agonistic activity (FIG. 3B). The fluctuation in activity due to
introduction of the P331S variation was not observed.
Example 9
Measurement of CDC Activity of Anti-CD40 Agonistic Antibody 331Ser
Mutant
[0163] CDC activity was measured by the above method. A rabbit
serum-derived complement was used, and Ramos cells were used as
target cells. The results confirmed that, in a KM341-1-19 antibody
at an antibody concentration of 1 .mu.g/ml, IgG2ser exhibited CDC
activity significantly reduced as compared with IgG2 (FIG. 4A). On
the other hand, when a human supplement was used, no change was
observed (FIG. 4B).
Example 10
Preparation and Activity Measurement of Agonist Anti-CD40 Antibody
Having Constant Region Converted
[0164] Among the anti-CD antibodies described in WO 02/088186, two
antibodies exhibiting strongest agonistic activity (KM341-1-19
antibody and 2105 antibody) belong to IgG2 subclass. In order to
examine whether or not the IgG2 subclass is important for
activation of CD40, recombinant proteins having an antibody
constant region converted into IgG1, IgG3 and IgG4, respectively,
were prepared, and measured in terms of binding ability to an
antigen and CD95 expression enhancing activity in Ramos cells
according to Examples 4 and 6. IgG1 was expressed using N5KG1, and
IgG2 and IgG3 were respectively expressed using expression vectors
N5KG2 and N5KG3 obtained by substituting the N5KG1 constant region
with IgG2 and IgG3, respectively. cDNA cloning of the IgG3 constant
region was carried out according to the IgG2 cloning method
partially modified, using an IgG3-specific primer. IgG4 was
expressed using N5KG4PE (IDEC Pharmaceuticals).
[0165] The antibody protein was expressed according to Example 1.
Binding activity to Ramos cells expressing human CD40 of the
KM341-1-19 antibody and the 2105 antibody was not affected by
converting IgG2 into IgG1, IgG3 or IgG4 (FIGS. 5A-1 and 5A-2).
However, these antibodies were found to have CD95 expression
enhancing activity in Ramos cells reduced by 10% or more (FIGS.
5B-1 and 5B-2). This shows that not only the structure of the
variable region defining the binding region of the antibody but
also the structure of the constant region of the antibody are
important for the strong agonistic activity of the 2105 antibody
and the KM341-1-19 antibody. Thus, in order to examine which region
in the IgG2 constant region is important for agonistic activity, a
domain swap mutant in which the IgG2 structure is mixed with the
IgG4 structure was prepared to measure its activity. As described
below, a domain swap mutant is prepared by substitution of a hinge
region. In this case, the "hinge region" includes the upper hinge
(from Kabat EU code 216), middle hinge (from Kabat EU code 226) and
lower hinge (Kabat EU code 231), as described Ole H Brekke et. al.,
Immunology Today 1995, 16, 85-90. Four domain swap mutants IgG2/4
(CH1 and hinge region: IgG2, other regions: IgG4), IgG4/2/4 (hinge
region: IgG2, other regions: IgG4), IgG2/4/4 (CH1: IgG2, other
regions: IgG4) and IgG4/2/2 (CH1: IgG4, other regions: IgG2) were
prepared respectively for the KM341-1-19 antibody and the 2105
antibody.
[0166] A vector N5KG2/4 for expressing the IgG2/4 antibody was
prepared using a Ztaq PCR kit (Takara). Reaction at 98.degree. C.
for 1 second, at 60.degree. C. for 30 seconds and at 72.degree. C.
for 30 seconds was carried out 15 times using N5KG2 as a template
and primers IgG3Bam: atatggatccTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID
NO: 8) and 24Chi4: AGGGGTCCGGGAGATCATGAGAGTGTCCTT (SEQ ID NO: 9).
At the same time, reaction at 98.degree. C. for 1 second, at
60.degree. C. for 30 seconds and at 72.degree. C. for 30 seconds
was carried out 15 times using N5KG4 (IDEC Pharmaceuticals) as a
template and primers 24Chi3: AAGGACACTCTCATGATCTCCCGGACCCCT (SEQ ID
NO: 10) and linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 11). The
amplified DNA fragments were purified using a PCR purification kit,
and the same amounts of the two purified DNA fragments were mixed.
Thereafter, reaction at 98.degree. C. for 1 second, at 60.degree.
C. for 30 seconds and at 72.degree. C. for 30 seconds was carried
out five times. Primers IgG3Bam and linkH2: tgatcatacgtagatatcacggc
(SEQ ID NO: 12) were added to the mixture, and the same reaction
was carried out 15 times. The amplified DNA fragment was cleaved
with NheI and BamHI, and substituted with the IgG1 constant region
of the N5KG1 vector.
[0167] A vector N5KG4/2/4 for expressing IgG4/2/4 was prepared as
follows. Reaction at 98.degree. C. for 1 second, at 60.degree. C.
for 30 seconds and at 72.degree. C. for 30 seconds was carried out
15 times using N5KG4 as a template and primers linkH:
gggtacgtcctcacattcagtgatcag (SEQ ID NO: 13), G2Hin3:
TTTGCGCTCAACTGTCTTGTCCACCTTGGTGTTGCTGGG (SEQ ID NO: 14), linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 15) and G2Hin4:
ACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCG (SEQ ID NO: 16). The amplified
DNA fragments were purified with a PCR purification kit, and the
same amounts of the two purified DNA fragments were mixed.
Thereafter, reaction at 98.degree. C. for 1 second, at 60.degree.
C. for 30 seconds and at 72.degree. C. for 30 seconds was carried
out five times using the mixture as a template. Primers linkH and
linkH2 were added to the mixture, and the same reaction was carried
out 15 times. The amplified DNA fragment was cleaved with NheI and
BamHI, and substituted with the IgG1 constant region of the N5KG1
vector.
[0168] A vector N5KG2/4/4 for expressing IgG2/4/4 was prepared as
follows. Reaction at 98.degree. C. for 1 second, at 60.degree. C.
for 30 seconds and at 72.degree. C. for 30 seconds was carried out
15 times using N5KG2 as a template and primers linkH:
gggtacgtcctcacattcagtgatcag (SEQ ID NO: 17) and G4CH1-2:
GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 18). At the same time,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out 15
times using N5KG4 as a template and primers G4CH1-1:
CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 19) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 20). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1
constant region of the N5KG1 vector.
[0169] A vector N5KG4/2/2 for expressing IgG4/2/2 was prepared as
follows. Reaction at 98.degree. C. for 1 second, at 60.degree. C.
for 30 seconds and at 72.degree. C. for 30 seconds was carried out
using N5KG4 as a template and primers linkH:
gggtacgtcctcacattcagtgatcag (SEQ ID NO: 21) and G4CH1-2:
GGTGTTGCTGGGCTTGTGATCTACGTTGCAG (SEQ ID NO: 22). At the same time,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out 15
times using N5KG2 as a template and primers G4CH1-1:
CTGCAACGTAGATCACAAGCCCAGCAACACC (SEQ ID NO: 23) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 24). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1
constant region of the N5KG1 vector.
[0170] Binding activity of the respective four domain swap mutants
of the KM341-1-19 antibody and the 2105 antibody was examined. As a
result, no difference between them and the original IgG2 was
observed in terms of binding ability (FIGS. 6A-1 and 6A-2).
However, only IgG2/4/4 of both the KM341-1-19 antibody and the 2105
antibody exhibited significantly reduced agonistic activity (FIGS.
6B-1 and 6B-2). The results confirmed that the hinge region of IgG2
is important for agonistic activity.
[0171] Further, it was examined which sequence is important in the
hinge region. The hinge region is divided into three sites,
specifically, upper hinge, middle hinge and lower hinge (Ole H
Brekke et al. Immunology Today 1995, 16, 85-90). IgG2-specific
sequences in these regions were respectively substituted with
IgG4-specific sequences. Antibodies obtained by introducing a
mutation into the upper hinge (from Kabat EU code 216), middle
hinge (from Kabat EU code 226) and lower hinge (from Kabat EU code
231) were respectively defined as IgG2UH4, IgG2MH4 and IgG2LH4.
Their respective expression vectors were defined as N5KG2UH4,
N5KG2MH4 and N5KG2LH4. "Hinge" is defined as EU indices 216 to 230
according to Kabat et al., Sequences of proteins of immunological
interest, 1991 Fifth edition.
[0172] N5KG2UH4 was prepared as follows. Reaction at 98.degree. C.
for 1 second, at 60.degree. C. for 30 seconds and at 72.degree. C.
for 30 seconds was carried out 15 times using N5KG2 as a template
and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 25) and
UH4-2: CACAACATTTggaCTCAACTcTCTTGTCCACC (SEQ ID NO: 26). At the
same time, reaction at 98.degree. C. for 1 second, at 60.degree. C.
for 30 seconds and at 72.degree. C. for 30 seconds was carried out
15 times using N5KG2 as a template and primers UH4-1:
GGTGGACAAGAgAGTTGAGtccAAATGTTGTG (SEQ ID NO: 27) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 28). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1
constant region of the N5KG1 vector.
[0173] N5KG2MH4 was prepared as follows. Reaction at 98.degree. C.
for 1 second, at 60.degree. C. for 30 seconds and at 72.degree. C.
for 30 seconds was carried out 15 times using N5KG2 as a template
and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 29) and
UM4-2: GGCACGGTGGGCAtgggggaccataTTTGCGCTC (SEQ ID NO: 30). At the
same time, reaction at 98.degree. C. for 1 second, at 60.degree. C.
for 30 seconds and at 72.degree. C. for 30 seconds was carried out
15 times using N5KG2 as a template and primers UM4-1:
GAGCGCAAAtatggtcccccaTGCCCACCGTGCC (SEQ ID NO: 31) and linkH2:
tgatcatacgtagatatcacggc (SEQ ID NO: 32). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1
constant region of the N5KG1 vector.
[0174] N5KG2LH4 was prepared as follows. Reaction at 98.degree. C.
for 4 second, at 60.degree. C. for 30 seconds and at 72.degree. C.
for 30 seconds was carried out 15 times using N5KG2 as a template
and primers linkH: gggtacgtcctcacattcagtgatcag (SEQ ID NO: 33) and
UL4-2: GAAGACTGACGGTCCccccaggaactcTGGTGCTGGGCA (SEQ ID NO: 34). At
the same time, reaction at 98.degree. C. for 1 second, at
60.degree. C. for 30 seconds and at 72.degree. C. for 30 seconds
was carried out 15 times using N5KG2 as a template and primers
UL4-1: TGCCCAGCACCAgagttcctggggGGACCGTCAGTCTTC (SEQ ID NO: 35) and
linkH2: tgatcatacgtagatatcacggc (SEQ ID NO: 36). The amplified DNA
fragments were purified using a PCR purification kit, and the same
amounts of the two purified DNA fragments were mixed. Thereafter,
reaction at 98.degree. C. for 1 second, at 60.degree. C. for 30
seconds and at 72.degree. C. for 30 seconds was carried out five
times. Primers linkH and linkH2 were added to the mixture, and the
same reaction was carried out 15 times. The amplified DNA fragment
was cleaved with NheI and BamHI, and substituted with the IgG1
constant region of the N5KG1 vector.
[0175] The three respective domain swap mutants of the KM341-1-19
antibody and the 2105 antibody were examined to have the same
binding activity to an antigen (FIGS. 6A-1 and 6A-2). However,
IgG2UH4 and IgG2MH4 exhibited significantly reduced agonistic
activity to Ramos cells (FIGS. 6B-1 and 6B-2). It was found from
the above that the structures of upper hinge and middle hinge in
the hinge region are important for IgG2 subclass-dependent
agonistic activity of the anti-CD40 antibodies KM341-1-19 and
2105.
[0176] Since IgG2 subclass was found to be important for agonistic
activity, antibodies of subclass other than IgG2 were converted to
those of IgG2 subclass to examine whether or not the agonistic
activity was enhanced. In the examination on several clones,
agonistic activity of F76 could be enhanced by converting IgG1
subclass to IgG2 subclass (FIGS. 7A and B).
Example 11
Preparation of Anti-CD40 Antagonist Antibody Mutants
[0177] A DNA fragment containing a heavy chain and a light chain of
a 4D11 antibody gene described in WO 02/088186, whose original
subclass is IgG1, was digested with BglII and NheI, purified, and
then integrated into N5KG4PE, N5KG4P and N5KG4 vectors (IDEC
Pharmaceuticals). N5KG4PE contains point mutations S228P and L235E
in the IgG4 constant region, and N5KG4P contains a point mutation
S228P in the IgG4 constant region. The antibody protein was
expressed and purified according to the above method. The antibody
was purified according to the above method using binding to Ramos
cells as an index. Change in binding activity of IgG1, IgG4, IgG4P
and IgG4PE to Ramos cells was not observed (FIG. 8A). Antagonistic
activity of IgG1 was compared with those of various IgG4 mutants
according to the above method to find that antagonistic activity of
IgG1 does not differ from those of the IgG4 mutants (FIG. 8B).
Example 12
Evaluation of ADCC Activity and CDC Activity of Anti-CD40
Antagonist Antibody Mutants
[0178] ADCC activity and CDC activity of anti-CD40 mutant
antibodies were evaluated according to the above method.
[0179] When using human MNC as effector cells and CD40-expressing
Daudi cells as target cells, two mutants IgG4 and IgG4PE were
respectively observed to have ADCC activity significantly reduced
as compared with IgG1 as the original subclass of the 4D11 antibody
(FIG. 9).
[0180] CDC activity of IgG1 was compared with that of IgG4P using
Daudi cells as target cells. IgG4P was found to have CDC activity
significantly reduced as compared with IgG1 (FIG. 10).
Example 13
Effect of Anti-CD40 Antagonistic Antibody on B Cells
[0181] 100 .mu.g each of IgG1, IgG4P and IgG4PE of the 4D11
antibody was administered to the tail vein of mice having a genetic
background whereby they were homozygotes for mouse endogenous
disrupted CD40 and harboring a transgene of a human CD40 gene
(Yasui. et al. Int. Immunol. 2002 Vol 14: 319). 24 hours after the
administration, blood was collected from the orbital venous plexus.
After hemolysis with 0.16 mol/L of ammonium chloride, an
FITC-labeled anti-B220 antibody was added to the hemolysate, and it
was analyzed using FACS. The results are shown in FIG. 11. In the
figure, the longitudinal axis indicates the ratio of B cells in the
total lymphocytes. IgG1 reduced the ratio of B cells most, IgG4P
reduced the ratio to a lesser extent, and IgG4PE reduced the ratio
to a much lesser extent. 24 hours after the administration, the
spleen was removed and crushed with a slide glass to prepare a cell
suspension. After hemolysis of the cell suspension, a PE-labeled
anti-B220 antibody and an FITC-labeled anti-CD23, CD86 or CD95
antibody were used for the hemolysate, and it was analyzed using
FACS. The results are shown in FIGS. 12A, B and C. In the figures,
the longitudinal axis indicates the ratio of B cells expressing
each surface marker in the total lymphocytes. 4D11G1 was found to
achieve the same level of increase in expression of each marker as
in a commercially available mouse anti-human CD40 agonistic
antibody 5C3 (Pharmingen). IgG4PE achieved a smaller increase in
expression of each activation surface marker as compared with IgG1
and IgG4P.
Example 14
Effect of Inhibiting Production of Antigen-Specific Antibody and
Change in the Number of B Cells Caused by Anti-CD40 Antagonistic
Antibody
[0182] 100 .mu.g (based on NP-CGG) of a complex of
4-hydroxy-3-nitrophenylacetyl-chiken .gamma.-globulin conjugates
(NP-CGG: distributed by Professor Hitoshi KIKUTANI, Research
Institute for Microbial Diseases, Osaka University) and alum
(aluminum hydroxide gel) was intraperitoneally administered to mice
having a genetic background whereby they were homozygotes for mouse
endogenous disrupted CD40 and harboring a transgene of a human CD40
gene (Yasui et al. Int. Immunol. 2002 Vol 14: 319) to sensitize the
mice. Immediately before the antigen sensitization, 50 or 100 .mu.g
of each antibody was administered to the tail vein. 100 .mu.g of an
anti-human albumin human IgG4PE antibody was administered as a
negative control. 7 and 14 days after the sensitization, blood was
collected from the orbital venous plexus. The amounts of
NP-specific IgG1 and IgM antibodies in the serum were measured by
the ELISA method. The ELISA method was carried out as follows. 50
.mu.l/well of NP-bound bovine serum albumin (NP-BSA: 2.5 .mu.g/ml)
was added to each well of a 96-well microplate for ELISA (Maxisorp,
manufactured by Nunc A/S) and incubated at 4.degree. C. to cause
NP-BSA to be adsorbed thereon. Next, the supernatant was discarded,
and a blocking reagent (SuperBlock, manufactured by Pierce
Biotechnology, Inc.) was added to each well and incubated at room
temperature to carry out blocking. Then, each well was washed with
a phosphate buffer (PBS-T) containing 0.1% Tween 20 three times.
Next, each serum diluted with PBS-T containing 10% Block Ace (50
.mu.l/well) was added to each well, and incubated and reacted at
37.degree. C. for two hours. The microplate was washed with PBS-T
three times. Then, a 1,000-fold dilution of a goat anti-mouse IgG1
antibody or IgM antibody labeled with alkaline phosphatase (Cosmo
Bio, 1070-04 or 1020-04) with PBS-T containing 10% Block Ace (50
.mu.g/well) was added to each well, and incubated at 37.degree. C.
for two hours. Next, the microplate was washed with PBS-T, and then
a coloring substrate solution (50 .mu.l/well, Sigma 104,
phosphatase substrate) was added to each well. The absorbance at a
wavelength of 405 nm was measured using a microplate reader. The
results are shown in FIGS. 13A and B. In the figures, the
longitudinal axis indicates values obtained by converting a
10,000-fold dilution (in the case of IgG1) or 100-fold dilution (in
the case of the IgM antibody) of serum collected from C57BL/6 mice,
to which NP-CGG was injected twice, and pooled into one unit. The
4D11 antibody and the IgG4P or IgG4PE antibody of 281 inhibited
production of NP-specific IgG1 and IgM antibodies equally
strongly.
[0183] The change in the number of B cells in the peripheral blood
and spleen in the mice used for examining the effect of inhibiting
antibody production was measured according to the same method as in
Example 1. The results are shown in FIGS. 14A and B. The 4D11
antibody and the IgG4P antibody of 281 reduced the ratio of B cells
in peripheral blood significantly as compared with the IgG4PE
antibody. Administration of 100 .mu.g of the IgG4PE antibody did
not change the ratio of B cells in the spleen removed 14 days after
the antigen sensitization. However, administration of IgG4P changed
or tended to change the ratio.
Example 15
Effect of Anti-CD40 Antagonistic Antibody on Cynomolgus Monkeys
[0184] 30 mg/kg of IgG4P or IgG4PE of a 4D11 antibody was
administered to the forearm cephalic vein of cynomolgus monkeys,
and blood was collected from the femoral vein after a certain
period of time. In the subset analysis of peripheral blood
lymphocytes, an FITC-labeled anti-CD3 antibody, PE-labeled
anti-CD20 antibody and APC-labeled anti-CD45 antibody were used for
each cell suspension, and the ratio of positive cells was measured
using FACS to calculate the ratio of CD45 positive cells. The
results are shown in FIG. 15. In the figure, the longitudinal axis
indicates the ratio of CD20 positive cells at each time to CD20
positive cells before antibody administration. 1 to 7 days after
the antibody administration, CD20 positive cells were reduced by
about 40% in individuals to which the IgG4P antibody was
administered. However, 4 days after the administration, CD20
positive cells were reduced by only about 20% in individuals to
which the IgG4PE antibody was administered.
[0185] The IL12 concentration in serum was measured by the ELISA
method. Blood collected from the femoral vein was allowed to stand
at room temperature for 20 to 60 minutes, and then centrifuged at
3,000 rpm at room temperature for 15 minutes. The IL12
concentration in the resulting serum was measured using a monkey
IL12 ELISA kit (BioSource International Inc.). The results are
shown in FIG. 16. No increase in IL12 production by the IgG4PE
antibody was observed at any blood collection point. However,
maximum IL12 production by the IgG4P antibody was observed on the
4th day.
Example 16
Effect of Anti-CD40 Antagonistic Antibody on Cynomolgus Monkey
Delayed Hypersensitivity Model
[0186] Nine male cynomolgus monkeys were intradermally and
intramuscularly sensitized with Tetanus toxoid (TTx) (10 Lf/ml;
Denka Seiken Co., Ltd.) to induce delayed hypersensitivity to TTx.
At the same time, 10 minutes before the start of sensitization, 0.1
and 10 mg/kg of a 4D11G4PE antibody was intravenously administered
to each three animals three times (once a week) to examine the
effect of 4D11G4PE on delayed hypersensitivity. Under anesthesia by
intramuscular administration of ketamine, sensitization was carried
out by intradermal administration of TTx to the back (50
.mu.L/site.times.12 sites) and intramuscular administration of TTx
to the femur (0.6 mL/body), and challenge was carried out by
intradermal administration of TTx to the thorax (10 .mu.L/site, 0
to 10 Lf/ml for each three sites) 21 days after the sensitization.
24 and 48 hours after the elicitation, skin reaction at the
administration sites was observed and evaluated according to the
Draize skin irritation score. The results of determining the TTx
concentration in each three sites were respectively a mean value.
The results are shown in FIG. 17. Administration of the 4D11G4PE
antibody apparently inhibited the delayed hypersensitivity reaction
observed 24 and 48 hours after the administration.
[0187] The effect of TTx on TTx-specific IgG and IgM antibody
titers was examined Blood collected from the femoral vein over time
was allowed to stand at room temperature for 20 to 60 minutes, and
then centrifuged at 3,000 rpm at room temperature for 15 minutes.
The antibody titer in the resulting serum was measured using the
ELISA method. The ELISA method was carried out as follows. 100
.mu.l/well of TTx (0.5 Lf/ml) was added to each well of a 96-well
microplate for ELISA (Maxisorp, manufactured by Nunc A/S) and
incubated at 4.degree. C. to cause TTx to be adsorbed thereon.
Next, the supernatant was discarded, and a blocking reagent
(phosphate buffer containing 0.5% BSA) was added to each well and
incubated at room temperature to carry out blocking. Then, each
well was washed with a phosphate buffer (PBS-T) containing 0.05%
Tween 20 three times. Next, each serum diluted with PBS-T
containing 0.5% BSA (100 to 819,200-fold dilution, dilution
magnification: 2; 100 .mu.l/well) was added to each well, and
incubated and reacted at room temperature for two hours. The
microplate was washed with PBS-T three times. Then, a 3,000-fold
dilution of a goat anti-monkey IgG antibody or IgM antibody labeled
with peroxidase (Nordic Immunology) with PBS-T containing 0.5% BSA
(100 .mu.g/well) was added to each well, and incubated at room
temperature for one hour. Next, the microplate was washed with
PBS-T, and then a coloring substrate solution (100 .mu.l/well,
o-phenylenediamine hydrochloride+aqueous hydrogen peroxide) was
added to each well. The absorbance at a wavelength of 492 nm was
measured using a microplate reader. The anti-TTx antibody titer was
defined as a maximum dilution magnification to make the absorbance
0.1 and more. The antibody titer was 0 when the absorbance did not
reach 0.1 even at 100-fold dilution. The results are shown in FIGS.
18 and 19. Administration of 1 mg/kg of 4D11G4PE suppressed the
TTx-specific IgG and IgM antibody titers to about 1/10. When 10
mg/kg of 4D11G4PE was administered, the antibody titers were below
the detection sensitivity at any blood collection point.
Example 17
Effect of Anti-CD40 Antagonistic Antibody on Platelet Thrombus
Formation
[0188] Blood collected from a healthy human was divided into four
aliquots (each 6 ml). Control human IgG4PE, control mouse IgG2a,
human anti-human CD40 IgG4PE (4D11) and mouse anti-human CD154
IgG2a (5C8) were respectively added to the fractions so that each
fraction had a blood concentration of 100 .mu.g/ml. A flat
perfusion chamber (GlycoTech Corp.) and a collagen-coated Petri
dish were assembled according to the attached instruction. The
blood treated with various antibodies was caused to flow into the
chamber at a rate that can apply a shear stress of 1,500/s to the
blood for seven minutes. Thereafter, a 4% paraformaldehyde
phosphate buffer was caused to flow into the chamber at a rate that
can apply a shear stress of 1,500/s to the buffer for 10 minutes.
The platelet aggregate formed on the Petri dish was fixed, stained
with a platelet-specific PE-labeled CD41a antibody, and observed
with a fluorescence microscope. The results are shown in FIGS. 20A
and B. The blood treated with human anti-human CD40 IgG4PE (4D11)
formed a platelet aggregate on the collagen-coated Petri dish, as
the blood treated with the control antibodies did. However, the
blood treated with mouse anti-human CD154 IgG2a did not form a
platelet aggregate.
Example 18
Evaluation of Stability of Anti-CD40 Antagonistic Antibody
[0189] Constant region-modified antibodies of the 4D11 antibody
were compared and examined in terms of stability. In the evaluation
method, culture supernatants obtained by respectively transiently
expressing G4P, G4PE, G2Ser and G4/2/4 in HEK293 cells were charged
with a Protein A column (Amersham Pharmacia Biotech), eluted with a
0.1 M citrate buffer (pH 2.7), and then incubated at 37.degree. C.
for 1 minute and 10 minutes. Thereafter, they are neutralized with
a 50 nM phosphate buffer (pH 7.0). The oligomer content in the
resulting antibody solutions was measured using a gel filtration
column (Tosoh Corp.). As a result, it was found that the oligomer
content increases in proportion with the incubation time, and
G4/2/4 produces an oligomer easiest, G4PE second easiest, G2Ser
third easiest, and G4P fourth easiest (FIG. 21).
Example 19
Effect of Inhibiting Skin Graft Rejection by Anti-CD40 Antagonistic
Antibody
[0190] A graft collected from the tail of DBA/2 mice was grafted
into the side dorsal thorax of C57BL/6 background mice having a
genetic background whereby they were homozygotes for mouse
endogenous disrupted CD40 and harboring a transgene of a human CD40
gene, and the graft was fixed with a plaster for seven days. 100
.mu.g of a test substance 4D11G4PE or a vehicle was administered to
the tail vein 0, 2, 4, 7, 9, 11 and 14 days after the skin graft,
respectively. To inhibit graft rejection by NK cells, 100 .mu.g of
an anti-asialo GML antibody was intraperitoneally administered to
all mice 3 days before the operation and 1, 4 and 9 days after the
operation. The results are shown in FIG. 22. The delay in graft
rejection was observed to be significant in the
4D11G4PE-administered group as compared with the
vehicle-administered group.
Example 20
Analysis of CD40 Expression in Human Tumor Cell Lines
[0191] Expression of CD40 in a Burkitt's lymphoma cell line Ramos,
bladder cancer cell line T24 (ATCC, HTB-4), pancreatic cancer cell
line Hs 766T (ATCC, HTB-134) and Capan-2 (ATCC, HTB-80) was
confirmed by FACS analysis using 341G2Ser.
[0192] T24, Hs 766T and Capan-2 were digested with trypsin and
harvested, and Ramos was harvested as is. The cell lines were
washed with PBS, and then re-suspended in a staining buffer
containing 1 .mu.g/ml of 341G2Ser. The staining buffer was prepared
by adding 0.05 mM EDTA, 0.05% sodium azide and 5% immobilized
bovine serum to PBS. After incubation at 4.degree. C. for 15
minutes, the cells were washed with the staining buffer twice, and
re-suspended in a 1:250 dilution of PE-bound goat anti-human IgG
(.gamma.) (Southern Biotechnology Associates, Inc) with the
staining buffer. After incubation at 4.degree. C. for 15 minutes,
the cells were washed with the staining buffer twice, and analyzed
with FACSCalibur.TM. (manufactured by BD Biosciences). The same
amount of a human anti-2,4-dinitrophenol (DNP) antibody was used as
a negative control. The analysis was carried out using
Cellquest.TM. (manufactured by BD Biosciences) as data analysis
software to calculate the mean fluorescence intensity.
[0193] As a result, Ramos, T24, Hs766T and Capan-2 had a mean
fluorescence intensity obviously higher than that of the negative
control when stained with 341G2Ser, and thus expression of CD40 was
confirmed.
Example 21
Effect of Anti-CD40 Agonistic Antibody on Human Tumor Cell
Lines
[0194] 2.5.times.10.sup.3 Ramos cells, 2.5.times.10.sup.2 T24
cells, 5.times.10.sup.3 Hs766T cells and 5.times.10.sup.3 Capan-2
cells were respectively suspended in a medium to make the total
volume of 100 .mu.L in a flat bottom 96-well plate (manufactured by
Falcon). Ramos and Hs766T, Capan-2 and T24 were cultured for 66
hours, 90 hours and 114 hours, respectively, together with 341G2Ser
at a concentration of 1 ng/ml to 1,000 ng/ml at 37.degree. C. in
the presence of 5% CO.sub.2. 10 .mu.L (3.7 MBq/mL) of
.sup.3H-labeled thymidine (manufactured by Amersham Biosciences)
was added and cultured at 37.degree. C. in the presence of 5%
CO.sub.2 for six hours. Ramos cells were harvested on Printed
Filtermat A (manufactured by PerkinElmer, Inc.) using a 96 micro
cell harvester (manufactured by Skatron Instruments, Inc.), and
covered with a sample bag (manufactured by PerkinElmer, Inc.). 12
mL of Betaplate Scint (manufactured by PerkinElmer, Inc.) was
added, and the .beta.-ray dose was measured with a liquid
scintillation counter (Pharmacia 1205 Betaplate: manufactured by
Pharmacia Corp.). Hs 766T cells, T24 cells and Capan-2 cells were
respectively harvested on Unifilter (manufactured by PerkinElmer,
Inc.) using a harvester (manufactured by PerkinElmer, Inc.). A
special seal was attached to the back of each filter, and 20
.mu.L/well of MicroScint 20 (manufactured by PerkinElmer, Inc.) was
added thereto. The .beta.-ray dose was measured with a
scintillation counter (TopCount: manufactured by Packard Instrument
Co., Inc.). Data were expressed as cell survival rates (%) obtained
by dividing a mean of triplicate measurements obtained in three
independent tests by a value of non-treatment control.
[0195] As a result, the cell survival rates were reduced in all
cell lines depending on the 341G2Ser concentration (Table 1). When
adding 100 ng/ml of 341G2Ser, the Ramos cell survival rate was 58%,
the T24 cell survival rate was 22%, the Hs 766T cell survival rate
was 15%, and the Capan-2 cell survival rate was 77%. 341G2Ser was
found to have activity of inhibiting growth of Ramos cells, T24
cells, Hs 766T cells and Capan-2 cells.
TABLE-US-00025 TABLE 1 Cell survival rate 341G2Ser concentration
(ng/ml) Cell line 1 10 100 1000 Ramos 98.49% 81.68% 57.77% 55.26%
T24 97.94% 50.72% 21.97% 25.35% Hs 766T 34.67% 21.50% 14.67% 15.18%
Capan-2 100.94% 85.34% 76.76% 72.89%
Example 22
Effect of Anti-CD40 Agonistic Antibody on Mouse Cancer-Bearing
Model
(1) Ramos Cells
[0196] Six-week-old female Balb/c nude mice (purchased from CLEA
Japan, Inc.) were irradiated with 3Gy radiation, and
2.times.10.sup.7 cells/mouse of Ramos cells were subcutaneously
grafted into the back thereof 16 days after the graft, the size of
tumors that took there was measured. Cancer-bearing mice having a
tumor size of 50 to 170 mm.sup.3 were classified into groups each
consisting of five mice. 100 .mu.g/mouse of 341G2Ser (a solution in
200 .mu.l of PBS containing 1% nude mouse serum) was intravenously
administered to the cancer-bearing mice once on the 16th day, and
the tumor size was measured until the 47th day. A human anti-human
serum albumin (HAS) antibody was used as a negative control.
(2) T24 Cells
[0197] A T24 cell mass that had undergone subcutaneous passage in
the back of nude mice three times were removed, and subcutaneously
grafted into the back of six-week-old female Balb/c nude mice
(purchased from CLEA Japan, Inc.). The tumor cell mass to be
grafted is appropriately about 3 mm square. 10 days after the
graft, the size of engrafted tumors was measured. Cancer-bearing
mice having a tumor size of 80 to 200 mm.sup.3 were classified into
groups each consisting of five mice. 100 .mu.g/mouse of 341G2Ser (a
solution in 200 .mu.l of PBS containing 1% nude mouse serum) was
intravenously administered to the cancer-bearing mice once on the
10th day, and the tumor size was measured until the 29th day. The
same amount of a human anti-DNP antibody was used as a negative
control.
(3) Hs 766T Cells
[0198] 7.times.10.sup.5 cells/mouse of Hs 766T cells were
subcutaneously grafted into the back of eight-week-old female
Balb/c nude mice (purchased from CLEA Japan, Inc.). 16 days after
the graft, the size of engrafted tumors was measured.
Cancer-bearing mice having a tumor size of 50 to 140 mm.sup.3 were
classified into groups each consisting of five mice. 100
.mu.g/mouse of 341G2Ser (a solution in 200 .mu.l of PBS containing
1% nude mouse serum) was intravenously administered to the
cancer-bearing mice once on the 16th day, and the tumor size was
measured until the 32nd day. The same amount of a human anti-DNP
antibody was used as a negative control.
(4) Capan-2 Cells
[0199] 2.times.10.sup.6 cells/mouse of Capan-2 cells were
subcutaneously grafted into the back of six-week-old female Balb/c
nude mice (purchased from CLEA Japan, Inc.). 13 days after the
graft, the size of engrafted tumors was measured. Cancer-bearing
mice having a tumor size of 30 to 130 mm.sup.3 were classified into
groups each consisting of five mice. 10 or 100 .mu.g/mouse of
341G2Ser (a solution in 200 .mu.l of PBS containing 1% nude mouse
serum) was intravenously administered to the cancer-bearing mice
twice a week from the 13th day, and the tumor size was measured
until the 34th day. A human polyclonal antibody (hIgG)
(manufactured by Sigma Co.) was used as a negative control.
[0200] The tumor growth inhibition ratio (TGIR) was calculated from
the following formula. 100-[{(mean tumor volume of
341G2Ser-administered group on the last measurement day-mean tumor
volume of 341G2Ser-administered group on the day of start of
antibody administration)/(mean tumor volume of negative
control-administered group on the last measurement day-mean tumor
volume of negative control-administered group on the day of start
of antibody administration)}.times.100]
[0201] As a result, TGIR exceeded 100% in the T24, Hs766T and
Capan-2 cancer-bearing mice, and a decrease in the tumor volume was
observed in the mice. On the other hand, TGIR was 73.4% in the
Ramos cancer-bearing mice, and an increase in the tumor volume was
considerably suppressed in the mice (Table 2). FIGS. 23 to 26
respectively show the change in the tumor volume of cancer-bearing
mice to which Ramos cells, T24 cells, Hs 766T cells and Capan-2
cells were respectively engrafted.
TABLE-US-00026 TABLE 2 Tumor growth inhibition ratio Amount of
341G2Ser administered Cell line 10 .mu.g/head 100 .mu.g/head Ramos
-- 73.40% T24 -- 109.05% Hs 766T -- 108.55% Capan-2 103.49%
119.20%
[0202] The inhibition ratio in each cell line is a value on the
last measurement day.
INDUSTRIAL APPLICABILITY
[0203] As shown in the Examples, the anti-CD40 antibody of the
present invention having a constant region into which a mutation is
introduced and an anti-CD40 antibody in which a part of the
structure of the subclass is substituted with that of another
subclass have reduced ADCC activity and CDC activity, while
maintaining its activity. Accordingly, the antibody of the present
invention has the reduced cytotoxicity to CD40-expressing cells
when administered to a subject as a therapeutic antibody, and thus
can be used with safety.
[0204] All publications, patents and patent applications cited in
this specification are herein incorporated by reference in their
entirety.
Sequence Listing Free Text
SEQ ID NOS: 2 to 36: Synthetic DNAs
[0205] SEQ ID NOS: 49 to 130: Synthetic peptides
Sequence CWU 1
1
1421175PRTHomo sapiens 1Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr
Leu Ile Asn Ser Gln1 5 10 15Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys
Leu Val Ser Asp Cys Thr 20 25 30Glu Phe Thr Glu Thr Glu Cys Leu Pro
Cys Gly Glu Ser Glu Phe Leu 35 40 45Asp Thr Trp Asn Arg Glu Thr His
Cys His Gln His Lys Tyr Cys Asp 50 55 60Pro Asn Leu Gly Leu Arg Val
Gln Gln Lys Gly Thr Ser Glu Thr Asp65 70 75 80Thr Ile Cys Thr Cys
Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys 85 90 95Glu Ser Cys Val
Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys 100 105 110Gln Ile
Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val 115 120
125Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp
130 135 140Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly
Thr Asn145 150 155 160Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg
Leu Arg Ala Leu 165 170 175239DNAArtificial SequenceDescription of
Artificial SequenceSynthetic DNA 2atatgctagc accaagggcc catcggtctt
ccccctggc 39338DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 3atatggatcc tcatttaccc ggagacaggg agaggctc
38440DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 4atatgctagc accaagggcc catcggtctt ccccctggcg
40526DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 5gttttctcga tggaggctgg gaggcc
26638DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 6atatggatcc tcatttaccc ggagacaggg agaggctc
38726DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 7ggcctcccag cctccatcga gaaaac
26836DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 8atatggatcc tcatttaccc ggagacaggg agaggc
36930DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 9aggggtccgg gagatcatga gagtgtcctt
301030DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 10aaggacactc tcatgatctc ccggacccct
301123DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 11tgatcatacg tagatatcac ggc
231223DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 12tgatcatacg tagatatcac ggc
231327DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 13gggtacgtcc tcacattcag tgatcag
271439DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 14tttgcgctca actgtcttgt ccaccttggt gttgctggg
391523DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 15tgatcatacg tagatatcac ggc
231636DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 16acagttgagc gcaaatgttg tgtcgagtgc ccaccg
361727DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 17gggtacgtcc tcacattcag tgatcag
271831DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 18ggtgttgctg ggcttgtgat ctacgttgca g
311931DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 19ctgcaacgta gatcacaagc ccagcaacac c
312023DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 20tgatcatacg tagatatcac ggc
232127DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 21gggtacgtcc tcacattcag tgatcag
272231DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 22ggtgttgctg ggcttgtgat ctacgttgca g
312331DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 23ctgcaacgta gatcacaagc ccagcaacac c
312423DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 24tgatcatacg tagatatcac ggc
232527DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 25gggtacgtcc tcacattcag tgatcag
272632DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 26cacaacattt ggactcaact ctcttgtcca cc
322732DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 27ggtggacaag agagttgagt ccaaatgttg tg
322823DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 28tgatcatacg tagatatcac ggc
232927DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 29gggtacgtcc tcacattcag tgatcag
273034DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 30ggcacggtgg gcatggggga ccatatttgc gctc
343134DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 31gagcgcaaat atggtccccc atgcccaccg tgcc
343223DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 32tgatcatacg tagatatcac ggc
233327DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 33gggtacgtcc tcacattcag tgatcag
273439DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 34gaagactgac ggtcccccca ggaactctgg tgctgggca
393539DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 35tgcccagcac cagagttcct ggggggaccg tcagtcttc
393623DNAArtificial SequenceDescription of Artificial
SequenceSynthetic DNA 36tgatcatacg tagatatcac ggc 23371480DNAHomo
sapiens 37gtcgacgctg aattctggct gaccagggca gccaccagag ctccagacaa
tgtctgtctc 60cttcctcatc ttcctgcccg tgctgggcct cccatggggt gtcctgtcac
aggtccaact 120gcagcagtca ggtccaggac tggtgaagcc ctcgcagacc
ctctcactca cctgtgccat 180ctccggggac agtgtctcta gcaacagtgc
tacttggaac tggatcaggc agtccccatc 240gagagacctt gagtggctgg
gaaggacata ctacaggtcc aagtggtatc gtgattatgt 300aggatctgtg
aaaagtcgaa taatcatcaa cccagacaca tccaacaacc agttctccct
360gcagctgaac tctgtgactc ccgaggacac ggctatatat tactgtacaa
gagcacagtg 420gctgggaggg gattacccct actactacag tatggacgtc
tggggccaag ggaccacggt 480caccgtctct tcagcctcca ccaagggccc
atcggtcttc cccctggcgc cctgctccag 540gagcacctcc gagagcacag
cggccctggg ctgcctggtc aaggactact tccccgaacc 600ggtgacggtg
tcgtggaact caggcgctct gaccagcggc gtgcacacct tcccagctgt
660cctacagtcc tcaggactct actccctcag cagcgtggtg accgtgccct
ccagcaactt 720cggcacccag acctacacct gcaacgtaga tcacaagccc
agcaacacca aggtggacaa 780gacagttgag cgcaaatgtt gtgtcgagtg
cccaccgtgc ccagcaccac ctgtggcagg 840accgtcagtc ttcctcttcc
ccccaaaacc caaggacacc ctcatgatct cccggacccc 900tgaggtcacg
tgcgtggtgg tggacgtgag ccacgaagac cccgaggtcc agttcaactg
960gtacgtggac ggcgtggagg tgcataatgc caagacaaag ccacgggagg
agcagttcaa 1020cagcacgttc cgtgtggtca gcgtcctcac cgttgtgcac
caggactggc tgaacggcaa 1080ggagtacaag tgcaaggtct ccaacaaagg
cctcccagcc cccatcgaga aaaccatctc 1140caaaaccaaa gggcagcccc
gagaaccaca ggtgtacacc ctgcccccat cccgggagga 1200gatgaccaag
aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat
1260cgccgtggag tgggagagca atgggcagcc ggagaacaac tacaagacca
cacctcccat 1320gctggactca gacggctcct tcttcctcta cagcaagctc
accgtggaca agagcaggtg 1380gcagcagggg aacgtcttct catgctccgt
gatgcatgag gctctgcaca accactacac 1440gcagaagagc ctctccctgt
ctccgggtaa atgaggatcc 148038474PRTHomo sapiens 38Met Ser Val Ser
Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp1 5 10 15Gly Val Leu
Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val 20 25 30Lys Pro
Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser 35 40 45Val
Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro Ser 50 55
60Arg Asp Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr65
70 75 80Arg Asp Tyr Val Gly Ser Val Lys Ser Arg Ile Ile Ile Asn Pro
Asp 85 90 95Thr Ser Asn Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr
Pro Glu 100 105 110Asp Thr Ala Ile Tyr Tyr Cys Thr Arg Ala Gln Trp
Leu Gly Gly Asp 115 120 125Tyr Pro Tyr Tyr Tyr Ser Met Asp Val Trp
Gly Gln Gly Thr Thr Val 130 135 140Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala145 150 155 160Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 165 170 175Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly 180 185 190Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser 195 200
205Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe
210 215 220Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser
Asn Thr225 230 235 240Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys
Val Glu Cys Pro Pro 245 250 255Cys Pro Ala Pro Pro Val Ala Gly Pro
Ser Val Phe Leu Phe Pro Pro 260 265 270Lys Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 290 295 300Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu305 310 315
320Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val
325 330 335His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 340 345 350Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Thr Lys Gly 355 360 365Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 370 375 380Met Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr385 390 395 400Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440
445Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
450 455 460Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys465
47039406DNAHomo sapiens 39actgctcagt taggacccag agggaaccat
ggaagcccca gctcagcttc tcttcctcct 60gctactctgg ctcccagata ccaccggaga
aattgtgttg acacagtctc cagccaccct 120gtctttgtct ccaggggaaa
gagccaccct ctcctgcagg gccagtcaga gtgttagcag 180ctacttagcc
tggtaccaac agaaacctgg ccaggctccc aggctcctca tctatgatgc
240atccaacagg gccactggca tcccagccag gttcagtggc agtgggtctg
ggacagactt 300cactctcacc atcagcagcc tagagcctga agattttgca
gtttattact gtcagcagcg 360tagcaacact ttcggccctg ggaccaaagt
ggatatcaaa cgtacg 40640126PRTHomo sapiens 40Met Glu Ala Pro Ala Gln
Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Ser Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg Leu
Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser 100 105 110Asn Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg
Thr 115 120 12541508DNAHomo sapiens 41ctgaacacag acccgtcgac
tcccaggtgt ttccattcag tgatcagcac tgaacacaga 60ggactcacca tggagttggg
actgagctgg attttccttt tggctatttt aaaaggtgtc 120cagtgtgaag
tgcagctggt ggagtctggg ggaggcttgg tacagcctgg caggtccctg
180agactctcct gtgcagcctc tggattcacc tttgatgatt atgccatgca
ctgggtccgg 240caagctccag ggaagggcct ggagtgggtc tcaggtatta
gttggaatag tggtagcttg 300gtgcatgcgg actctgtgaa gggccgattc
accatctcca gagacaacgc caagaactcc 360ctgtatctgc aaatgaacag
tctgagagct gaggacacgg ccttgtatta ctgtgcaaga 420gataggctat
ttcggggagt taggtactac ggtatggacg tctggggcca agggaccacg
480gtcaccgtct cctcagctag caccaagg 50842146PRTHomo sapiens 42Met Glu
Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu Lys Gly1 5 10 15Val
Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25
30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu 50 55 60Glu Trp Val Ser Gly Ile Ser Trp Asn Ser Gly Ser Leu Val
His Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Leu 100 105 110Tyr Tyr Cys Ala Arg Asp Arg Leu Phe
Arg Gly Val Arg Tyr Tyr Gly 115 120 125Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140Thr
Lys14543414DNAHomo sapiens 43ctgctcagtt aggacccaga gggaaccatg
gaagccccag ctcagcttct cttcctcctg 60ctactctggc tcccagatac caccggagaa
attgtgttga cacagtctcc agccaccctg 120tctttgtctc caggggaaag
agccaccctc tcctgcaggg ccagtcagag tgttagcagc 180tacttagcct
ggtaccaaca gaaacctggc caggctccca ggctcctcat ctatgatgca
240tccaacaggg ccactggcat cccagccagg ttcagtggca gtgggtctgg
gacagacttc 300actctcacca tcagcagcct agagcctgaa gattttgcag
tttattactg tcagcagcgt 360agccactggc tcactttcgg cggggggacc
aaggtggaga tcaaacgtac ggtg 41444129PRTHomo sapiens 44Met Glu Ala
Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr
Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30Leu
Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40
45Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro
Ala65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser 85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser 100 105 110His Trp Leu Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys Arg Thr 115 120 125Val45462DNAHomo sapiens
45atatgtcgac gagtcatgga tctcatgtgc aagaaaatga agcacctgtg gttcttcctc
60ctgctggtgg cggctcccag atgggtcctg tcccagctgc agctgcagga gtcgggccca
120ggactactga agccttcgga gaccctgtcc ctcacctgca ctgtctctgg
cggctccatc 180agcagtcctg gttactacgg gggctggatc cgccagcccc
cagggaaggg gctggagtgg 240attgggagta tctataaaag tgggagcacc
taccacaacc cgtccctcaa gagtcgagtc 300accatatccg tagacacgtc
caagaaccag ttctccctga agctgagctc tgtgaccgcc 360gcagacacgg
ctgtgtatta ctgtacgaga cctgtagtac gatattttgg gtggttcgac
420ccctggggcc agggaaccct ggtcaccgtc tcctcagcta gc 46246149PRTHomo
sapiens 46Met Asp Leu Met Cys Lys Lys Met Lys His Leu Trp Phe Phe
Leu Leu1 5 10 15Leu Val Ala Ala Pro Arg Trp Val Leu Ser Gln Leu Gln
Leu Gln Glu 20 25 30Ser Gly Pro Gly Leu Leu Lys Pro Ser Glu Thr Leu
Ser Leu Thr Cys 35 40 45Thr Val Ser Gly Gly Ser Ile Ser Ser Pro Gly
Tyr Tyr Gly Gly Trp 50 55 60Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile Gly Ser Ile Tyr65 70
75 80Lys Ser Gly Ser Thr Tyr His Asn Pro Ser Leu Lys Ser Arg Val
Thr 85 90 95Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu
Ser Ser 100 105 110Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr
Arg Pro Val Val 115 120 125Arg Tyr Phe Gly Trp Phe Asp Pro Trp Gly
Gln Gly Thr Leu Val Thr 130 135 140Val Ser Ser Ala
Ser14547448DNAHomo sapiens 47agatcttaag caagtgtaac aactcagagt
acgcggggag acccactcag gacacagcat 60ggacatgagg gtccccgctc agctcctggg
gcttctgctg ctctggctcc caggtgccag 120atgtgccatc cagttgaccc
agtctccatc ctccctgtct gcatctgtag gagacagagt 180caccatcact
tgccgggcaa gtcagggcat tagcagtgct ttagcctggt atcagcagaa
240accagggaaa gctcctaagc tcctgatcta tgatgcctcc aatttggaaa
gtggggtccc 300atcaaggttc agcggcagtg gatctgggac agatttcact
ctcaccatca gcagcctgca 360gcctgaagat tttgcaactt attactgtca
acagtttaat agttacccga cgttcggcca 420agggaccaag gtggaaatca aacgtacg
44848130PRTHomo sapiens 48Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Ala Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile
Tyr Asp Ala Ser Asn Leu Glu Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Phe
Asn Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
125Arg Thr 1304913PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 49Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln
Tyr Leu Ile1 5 105013PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 50Pro Thr Ala Cys Arg Glu Lys
Gln Tyr Leu Ile Asn Ser1 5 105113PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 51Ala Cys Arg Glu Lys Gln
Tyr Leu Ile Asn Ser Gln Cys1 5 105213PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 52Arg
Glu Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys Ser1 5
105313PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 53Lys Gln Tyr Leu Ile Asn Ser Gln Cys Cys
Ser Leu Cys1 5 105413PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 54Tyr Leu Ile Asn Ser Gln Cys
Cys Ser Leu Cys Gln Pro1 5 105513PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 55Ile Asn Ser Gln Cys Cys
Ser Leu Cys Gln Pro Gly Gln1 5 105613PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 56Ser
Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu1 5
105713PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 57Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys
Leu Val Ser1 5 105813PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 58Ser Leu Cys Gln Pro Gly Gln
Lys Leu Val Ser Asp Cys1 5 105913PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 59Cys Gln Pro Gly Gln Lys
Leu Val Ser Asp Cys Thr Glu1 5 106013PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 60Pro
Gly Gln Lys Leu Val Ser Asp Cys Thr Glu Phe Thr1 5
106113PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 61Gln Lys Leu Val Ser Asp Cys Thr Glu Phe
Thr Glu Thr1 5 106213PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 62Leu Val Ser Asp Cys Thr Glu
Phe Thr Glu Thr Glu Cys1 5 106313PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 63Ser Asp Cys Thr Glu Phe
Thr Glu Thr Glu Cys Leu Pro1 5 106413PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 64Cys
Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly1 5
106513PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 65Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys
Gly Glu Ser1 5 106613PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 66Thr Glu Thr Glu Cys Leu Pro
Cys Gly Glu Ser Glu Phe1 5 106713PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 67Thr Glu Cys Leu Pro Cys
Gly Glu Ser Glu Phe Leu Asp1 5 106813PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 68Cys
Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp1 5
106913PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 69Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr
Trp Asn Arg1 5 107013PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 70Gly Glu Ser Glu Phe Leu Asp
Thr Trp Asn Arg Glu Thr1 5 107113PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 71Ser Glu Phe Leu Asp Thr
Trp Asn Arg Glu Thr His Cys1 5 107213PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 72Phe
Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln1 5
107313PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 73Asp Thr Trp Asn Arg Glu Thr His Cys His
Gln His Lys1 5 107413PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 74Trp Asn Arg Glu Thr His Cys
His Gln His Lys Tyr Cys1 5 107513PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 75Arg Glu Thr His Cys His
Gln His Lys Tyr Cys Asp Pro1 5 107613PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 76Thr
His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu1 5
107713PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 77Cys His Gln His Lys Tyr Cys Asp Pro Asn
Leu Gly Leu1 5 107813PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 78Gln His Lys Tyr Cys Asp Pro
Asn Leu Gly Leu Arg Val1 5 107913PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 79Lys Tyr Cys Asp Pro Asn
Leu Gly Leu Arg Val Gln Gln1 5 108013PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 80Cys
Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly1 5
108113PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 81Pro Asn Leu Gly Leu Arg Val Gln Gln Lys
Gly Thr Ser1 5 108213PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 82Leu Gly Leu Arg Val Gln Gln
Lys Gly Thr Ser Glu Thr1 5 108313PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 83Leu Arg Val Gln Gln Lys
Gly Thr Ser Glu Thr Asp Thr1 5 108413PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 84Val
Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys1 5
108513PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 85Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile
Cys Thr Cys1 5 108613PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 86Gly Thr Ser Glu Thr Asp Thr
Ile Cys Thr Cys Glu Glu1 5 108713PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 87Ser Glu Thr Asp Thr Ile
Cys Thr Cys Glu Glu Gly Trp1 5 108813PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 88Thr
Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys1 5
108913PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 89Thr Ile Cys Thr Cys Glu Glu Gly Trp His
Cys Thr Ser1 5 109013PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 90Cys Thr Cys Glu Glu Gly Trp
His Cys Thr Ser Glu Ala1 5 109113PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 91Cys Glu Glu Gly Trp His
Cys Thr Ser Glu Ala Cys Glu1 5 109213PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 92Glu
Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys1 5
109313PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 93Trp His Cys Thr Ser Glu Ala Cys Glu Ser
Cys Val Leu1 5 109413PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 94Cys Thr Ser Glu Ala Cys Glu
Ser Cys Val Leu His Arg1 5 109513PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 95Ser Glu Ala Cys Glu Ser
Cys Val Leu His Arg Ser Cys1 5 109613PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 96Ala
Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro1 5
109713PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 97Glu Ser Cys Val Leu His Arg Ser Cys Ser
Pro Gly Phe1 5 109813PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 98Cys Val Leu His Arg Ser Cys
Ser Pro Gly Phe Gly Val1 5 109913PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 99Leu His Arg Ser Cys Ser
Pro Gly Phe Gly Val Lys Gln1 5 1010013PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 100Arg
Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala1 5
1010113PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 101Cys Ser Pro Gly Phe Gly Val Lys Gln
Ile Ala Thr Gly1 5 1010213PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 102Pro Gly Phe Gly Val Lys Gln
Ile Ala Thr Gly Val Ser1 5 1010313PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 103Phe Gly Val Lys Gln Ile
Ala Thr Gly Val Ser Asp Thr1 5 1010413PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 104Val
Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys1 5
1010513PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 105Gln Ile Ala Thr Gly Val Ser Asp Thr
Ile Cys Glu Pro1 5 1010613PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 106Ala Thr Gly Val Ser Asp Thr
Ile Cys Glu Pro Cys Pro1 5 1010713PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 107Gly Val Ser Asp Thr Ile
Cys Glu Pro Cys Pro Val Gly1 5 1010813PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 108Ser
Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe1 5
1010913PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 109Thr Ile Cys Glu Pro Cys Pro Val Gly
Phe Phe Ser Asn1 5 1011013PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 110Cys Glu Pro Cys Pro Val Gly
Phe Phe Ser Asn Val Ser1 5 1011113PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 111Pro Cys Pro Val Gly Phe
Phe Ser Asn Val Ser Ser Ala1 5 1011213PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 112Pro
Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu1 5
1011313PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 113Gly Phe Phe Ser Asn Val Ser Ser Ala
Phe Glu Lys Cys1 5 1011413PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 114Phe Ser Asn Val Ser Ser Ala
Phe Glu Lys Cys His Pro1 5 1011513PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 115Asn Val Ser Ser Ala Phe
Glu Lys Cys His Pro Trp Thr1 5 1011613PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 116Ser
Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys1 5
1011713PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 117Ala Phe Glu Lys Cys His Pro Trp Thr
Ser Cys Glu Thr1 5 1011813PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 118Glu Lys Cys His Pro Trp Thr
Ser Cys Glu Thr Lys Asp1 5 1011913PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 119Cys His Pro Trp Thr Ser
Cys Glu Thr Lys Asp Leu Val1 5 1012013PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 120Pro
Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln1 5
1012113PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 121Thr Ser Cys Glu Thr Lys Asp Leu Val
Val Gln Gln Ala1 5 1012213PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 122Cys Glu Thr Lys Asp Leu Val
Val Gln Gln Ala Gly Thr1 5 1012313PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 123Thr Lys Asp Leu Val Val
Gln Gln Ala Gly Thr Asn Lys1 5 1012413PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 124Asp
Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp1 5
1012513PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 125Val Val Gln Gln Ala Gly Thr Asn Lys
Thr Asp Val Val1 5 1012613PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 126Gln Gln Ala Gly Thr Asn Lys
Thr Asp Val Val Cys Gly1 5 1012713PRTArtificial SequenceDescription
of Artificial SequenceSynthetic peptide 127Ala Gly Thr Asn Lys Thr
Asp Val Val Cys Gly Pro Gln1 5 1012813PRTArtificial
SequenceDescription of Artificial SequenceSynthetic peptide 128Thr
Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg1 5
1012913PRTArtificial SequenceDescription of Artificial
SequenceSynthetic peptide 129Lys Thr Asp Val Val Cys Gly Pro Gln
Asp Arg Leu Arg1 5 1013013PRTArtificial SequenceDescription of
Artificial SequenceSynthetic peptide 130Asp Val Val Cys Gly Pro Gln
Asp Arg Leu Arg Ala Leu1 5 101311425DNAHomo sapiens 131atgtctgtct
ccttcctcat cttcctgccc gtgctgggcc tcccatgggg tgtcctgtca 60caggtccaac
tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc
120acctgtgcca tctccgggga cagtgtctct agcaacagtg ctacttggaa
ctggatcagg 180cagtccccat cgagagacct tgagtggctg ggaaggacat
actacaggtc caagtggtat 240cgtgattatg taggatctgt gaaaagtcga
ataatcatca acccagacac atccaacaac 300cagttctccc tgcagctgaa
ctctgtgact cccgaggaca cggctatata ttactgtaca 360agagcacagt
ggctgggagg ggattacccc tactactaca gtatggacgt ctggggccaa
420gggaccacgg tcaccgtctc ctcagctagc accaagggcc catcggtctt
ccccctggcg 480ccctgctcca ggagcacctc cgagagcaca gcggccctgg
gctgcctggt caaggactac 540ttccccgaac cggtgacggt gtcgtggaac
tcaggcgctc tgaccagcgg cgtgcacacc 600ttcccagctg tcctacagtc
ctcaggactc tactccctca gcagcgtggt gaccgtgccc 660tccagcaact
tcggcaccca gacctacacc tgcaacgtag atcacaagcc cagcaacacc
720aaggtggaca agacagttga gcgcaaatgt tgtgtcgagt gcccaccgtg
cccagcacca 780cctgtggcag gaccgtcagt cttcctcttc cccccaaaac
ccaaggacac cctcatgatc 840tcccggaccc ctgaggtcac gtgcgtggtg
gtggacgtga gccacgaaga ccccgaggtc 900cagttcaact ggtacgtgga
cggcgtggag gtgcataatg ccaagacaaa gccacgggag 960gagcagttca
acagcacgtt ccgtgtggtc agcgtcctca
ccgttgtgca ccaggactgg 1020ctgaacggca aggagtacaa gtgcaaggtc
tccaacaaag gcctcccagc ctccatcgag 1080aaaaccatct ccaaaaccaa
agggcagccc cgagaaccac aggtgtacac cctgccccca 1140tcccgggagg
agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctac
1200cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa
ctacaagacc 1260acacctccca tgctggactc cgacggctcc ttcttcctct
acagcaagct caccgtggac 1320aagagcaggt ggcagcaggg gaacgtcttc
tcatgctccg tgatgcatga ggctctgcac 1380aaccactaca cgcagaagag
cctctccctg tctccgggta aatga 1425132474PRTHomo sapiens 132Met Ser
Val Ser Phe Leu Ile Phe Leu Pro Val Leu Gly Leu Pro Trp1 5 10 15Gly
Val Leu Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val 20 25
30Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser
35 40 45Val Ser Ser Asn Ser Ala Thr Trp Asn Trp Ile Arg Gln Ser Pro
Ser 50 55 60Arg Asp Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys
Trp Tyr65 70 75 80Arg Asp Tyr Val Gly Ser Val Lys Ser Arg Ile Ile
Ile Asn Pro Asp 85 90 95Thr Ser Asn Asn Gln Phe Ser Leu Gln Leu Asn
Ser Val Thr Pro Glu 100 105 110Asp Thr Ala Ile Tyr Tyr Cys Thr Arg
Ala Gln Trp Leu Gly Gly Asp 115 120 125Tyr Pro Tyr Tyr Tyr Ser Met
Asp Val Trp Gly Gln Gly Thr Thr Val 130 135 140Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala145 150 155 160Pro Cys
Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu 165 170
175Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
180 185 190Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser 195 200 205Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Asn Phe 210 215 220Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp
His Lys Pro Ser Asn Thr225 230 235 240Lys Val Asp Lys Thr Val Glu
Arg Lys Cys Cys Val Glu Cys Pro Pro 245 250 255Cys Pro Ala Pro Pro
Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp 290 295
300Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu305 310 315 320Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Val 325 330 335His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 340 345 350Lys Gly Leu Pro Ala Ser Ile Glu
Lys Thr Ile Ser Lys Thr Lys Gly 355 360 365Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 370 375 380Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr385 390 395 400Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410
415Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe
420 425 430Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 435 440 445Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 450 455 460Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys465 470133696DNAHomo sapiens 133atggaagccc cagctcagct tctcttcctc
ctgctactct ggctcccaga taccaccgga 60gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 120ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 180ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
240aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 300gaagattttg cagtttatta ctgtcagcag cgtagcaaca
ctttcggccc tgggaccaaa 360gtggatatca aacgtacggt ggctgcacca
tctgtcttca tcttcccgcc atctgatgag 420cagttgaaat ctggaactgc
ctctgttgtg tgcctgctga ataacttcta tcccagagag 480gccaaagtac
agtggaaggt ggataacgcc ctccaatcgg gtaactccca ggagagtgtc
540acagagcagg acagcaagga cagcacctac agcctcagca gcaccctgac
gctgagcaaa 600gcagactacg agaaacacaa agtctacgcc tgcgaagtca
cccatcaggg cctgagctcg 660cccgtcacaa agagcttcaa caggggagag tgttga
696134231PRTHomo sapiens 134Met Glu Ala Pro Ala Gln Leu Leu Phe Leu
Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser 20 25 30Leu Ser Pro Gly Glu Arg Ala Thr
Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Ser Ser Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg Leu Leu Ile Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala65 70 75 80Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Glu
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser 100 105 110Asn
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala 115 120
125Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
130 135 140Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu145 150 155 160Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser 165 170 175Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser Thr Tyr Ser Leu 180 185 190Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu Lys His Lys Val 195 200 205Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 210 215 220Ser Phe Asn
Arg Gly Glu Cys225 2301351407DNAHomo sapiens 135atggagttgg
gactgagctg gattttcctt ttggctattt taaaaggtgt ccagtgtgaa 60gtgcagctgg
tggagtctgg gggaggcttg gtacagcctg gcaggtccct gagactctcc
120tgtgcagcct ctggattcac ctttgatgat tatgccatgc actgggtccg
gcaagctcca 180gggaagggcc tggagtgggt ctcaggtatt agttggaata
gtggtagctt ggtgcatgcg 240gactctgtga agggccgatt caccatctcc
agagacaacg ccaagaactc cctgtatctg 300caaatgaaca gtctgagagc
tgaggacacg gccttgtatt actgtgcaag agataggcta 360tttcggggag
ttaggtacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc
420tcctcagcta gcaccaaggg cccatcggtc ttccccctgg cgccctgctc
caggagcacc 480tccgagagca cagcggccct gggctgcctg gtcaaggact
acttccccga accggtgacg 540gtgtcgtgga actcaggcgc tctgaccagc
ggcgtgcaca ccttcccagc tgtcctacag 600tcctcaggac tctactccct
cagcagcgtg gtgaccgtgc cctccagcaa cttcggcacc 660cagacctaca
cctgcaacgt agatcacaag cccagcaaca ccaaggtgga caagacagtt
720gagcgcaaat gttgtgtcga gtgcccaccg tgcccagcac cacctgtggc
aggaccgtca 780gtcttcctct tccccccaaa acccaaggac accctcatga
tctcccggac ccctgaggtc 840acgtgcgtgg tggtggacgt gagccacgaa
gaccccgagg tccagttcaa ctggtacgtg 900gacggcgtgg aggtgcataa
tgccaagaca aagccacggg aggagcagtt caacagcacg 960ttccgtgtgg
tcagcgtcct caccgttgtg caccaggact ggctgaacgg caaggagtac
1020aagtgcaagg tctccaacaa aggcctccca gcctccatcg agaaaaccat
ctccaaaacc 1080aaagggcagc cccgagaacc acaggtgtac accctgcccc
catcccggga ggagatgacc 1140aagaaccagg tcagcctgac ctgcctggtc
aaaggcttct accccagcga catcgccgtg 1200gagtgggaga gcaatgggca
gccggagaac aactacaaga ccacacctcc catgctggac 1260tccgacggct
ccttcttcct ctacagcaag ctcaccgtgg acaagagcag gtggcagcag
1320gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta
cacgcagaag 1380agcctctccc tgtctccggg taaatga 1407136468PRTHomo
sapiens 136Met Glu Leu Gly Leu Ser Trp Ile Phe Leu Leu Ala Ile Leu
Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln 20 25 30Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 35 40 45Asp Asp Tyr Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 50 55 60Glu Trp Val Ser Gly Ile Ser Trp Asn Ser
Gly Ser Leu Val His Ala65 70 75 80Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Asn 85 90 95Ser Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Leu 100 105 110Tyr Tyr Cys Ala Arg
Asp Arg Leu Phe Arg Gly Val Arg Tyr Tyr Gly 115 120 125Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser 130 135 140Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr145 150
155 160Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro 165 170 175Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val 180 185 190His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser 195 200 205Ser Val Val Thr Val Pro Ser Ser Asn
Phe Gly Thr Gln Thr Tyr Thr 210 215 220Cys Asn Val Asp His Lys Pro
Ser Asn Thr Lys Val Asp Lys Thr Val225 230 235 240Glu Arg Lys Cys
Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 245 250 255Ala Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265
270Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
275 280 285His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu 290 295 300Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr305 310 315 320Phe Arg Val Val Ser Val Leu Thr Val
Val His Gln Asp Trp Leu Asn 325 330 335Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Gly Leu Pro Ala Ser 340 345 350Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 355 360 365Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 370 375 380Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val385 390
395 400Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro 405 410 415Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr 420 425 430Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser Val 435 440 445Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu 450 455 460Ser Pro Gly
Lys465137702DNAHomo sapiens 137atggaagccc cagctcagct tctcttcctc
ctgctactct ggctcccaga taccaccgga 60gaaattgtgt tgacacagtc tccagccacc
ctgtctttgt ctccagggga aagagccacc 120ctctcctgca gggccagtca
gagtgttagc agctacttag cctggtacca acagaaacct 180ggccaggctc
ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc
240aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag
cctagagcct 300gaagattttg cagtttatta ctgtcagcag cgtagccact
ggctcacttt cggcgggggg 360accaaggtgg agatcaaacg tacggtggct
gcaccatctg tcttcatctt cccgccatct 420gatgagcagt tgaaatctgg
aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480agagaggcca
aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag
540agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac
cctgacgctg 600agcaaagcag actacgagaa acacaaagtc tacgcctgcg
aagtcaccca tcagggcctg 660agctcgcccg tcacaaagag cttcaacagg
ggagagtgtt ga 702138233PRTHomo sapiens 138Met Glu Ala Pro Ala Gln
Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro1 5 10 15Asp Thr Thr Gly Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30Leu Ser Pro Gly
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser 35 40 45Val Ser Ser
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 55 60Arg Leu
Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala65 70 75
80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
85 90 95Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg
Ser 100 105 110His Trp Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg Thr 115 120 125Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Ser Asp Glu Gln Leu 130 135 140Lys Ser Gly Thr Ala Ser Val Val Cys
Leu Leu Asn Asn Phe Tyr Pro145 150 155 160Arg Glu Ala Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170 175Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 180 185 190Ser Leu
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 195 200
205Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
210 215 220Thr Lys Ser Phe Asn Arg Gly Glu Cys225 2301391425DNAHomo
sapiens 139atggatctca tgtgcaagaa aatgaagcac ctgtggttct tcctcctgct
ggtggcggct 60cccagatggg tcctgtccca gctgcagctg caggagtcgg gcccaggact
actgaagcct 120tcggagaccc tgtccctcac ctgcactgtc tctggcggct
ccatcagcag tcctggttac 180tacgggggct ggatccgcca gcccccaggg
aaggggctgg agtggattgg gagtatctat 240aaaagtggga gcacctacca
caacccgtcc ctcaagagtc gagtcaccat atccgtagac 300acgtccaaga
accagttctc cctgaagctg agctctgtga ccgccgcaga cacggctgtg
360tattactgta cgagacctgt agtacgatat tttgggtggt tcgacccctg
gggccaggga 420accctggtca ccgtctcctc agctagcacc aaggggccat
ccgtcttccc cctggcgccc 480tgctccagga gcacctccga gagcacagcc
gccctgggct gcctggtcaa ggactacttc 540cccgaaccgg tgacggtgtc
gtggaactca ggcgccctga ccagcggcgt gcacaccttc 600ccggctgtcc
tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc
660agcagcttgg gcacgaagac ctacacctgc aacgtagatc acaagcccag
caacaccaag 720gtggacaaga gagttgagtc caaatatggt cccccatgcc
caccatgccc agcacctgag 780ttcgaggggg gaccatcagt cttcctgttc
cccccaaaac ccaaggacac tctcatgatc 840tcccggaccc ctgaggtcac
gtgcgtggtg gtggacgtga gccaggaaga ccccgaggtc 900cagttcaact
ggtacgtgga tggcgtggag gtgcataatg ccaagacaaa gccgcgggag
960gagcagttca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca
ccaggactgg 1020ctgaacggca aggagtacaa gtgcaaggtc tccaacaaag
gcctcccgtc ctccatcgag 1080aaaaccatct ccaaagccaa agggcagccc
cgagagccac aggtgtacac cctgccccca 1140tcccaggagg agatgaccaa
gaaccaggtc agcctgacct gcctggtcaa aggcttctac 1200cccagcgaca
tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc
1260acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaggct
aaccgtggac 1320aagagcaggt ggcaggaggg gaatgtcttc tcatgctccg
tgatgcatga ggctctgcac 1380aaccactaca cacagaagag cctctccctg
tctctgggta aatga 1425140474PRTHomo sapiens 140Met Asp Leu Met Cys
Lys Lys Met Lys His Leu Trp Phe Phe Leu Leu1 5 10 15Leu Val Ala Ala
Pro Arg Trp Val Leu Ser Gln Leu Gln Leu Gln Glu 20 25 30Ser Gly Pro
Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys 35 40 45Thr Val
Ser Gly Gly Ser Ile Ser Ser Pro Gly Tyr Tyr Gly Gly Trp 50 55 60Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Ser Ile Tyr65 70 75
80Lys Ser Gly Ser Thr Tyr His Asn Pro Ser Leu Lys Ser Arg Val Thr
85 90 95Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser
Ser 100 105 110Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Thr Arg
Pro Val Val 115 120 125Arg Tyr Phe Gly Trp Phe Asp Pro Trp Gly Gln
Gly Thr Leu Val Thr 130 135 140Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro145 150 155 160Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 165 170 175Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 180 185 190Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 195 200
205Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
210 215 220Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys225 230 235 240Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
Pro Cys Pro Pro Cys 245 250 255Pro Ala Pro Glu Phe Glu Gly Gly Pro
Ser Val Phe
Leu Phe Pro Pro 260 265 270Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 275 280 285Val Val Val Asp Val Ser Gln Glu
Asp Pro Glu Val Gln Phe Asn Trp 290 295 300Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu305 310 315 320Glu Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345
350Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
355 360 365Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
Glu Glu 370 375 380Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr385 390 395 400Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 405 410 415Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn 435 440 445Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460Gln
Lys Ser Leu Ser Leu Ser Leu Gly Lys465 470141708DNAHomo sapiens
141atggacatga gggtccccgc tcagctcctg gggcttctgc tgctctggct
cccaggtgcc 60agatgtgcca tccagttgac ccagtctcca tcctccctgt ctgcatctgt
aggagacaga 120gtcaccatca cttgccgggc aagtcagggc attagcagtg
ctttagcctg gtatcagcag 180aaaccaggga aagctcctaa gctcctgatc
tatgatgcct ccaatttgga aagtggggtc 240ccatcaaggt tcagcggcag
tggatctggg acagatttca ctctcaccat cagcagcctg 300cagcctgaag
attttgcaac ttattactgt caacagttta atagttaccc gacgttcggc
360caagggacca aggtggaaat caaacgtacg gtggctgcac catctgtctt
catcttcccg 420ccatctgatg agcagttgaa atctggaact gcctctgttg
tgtgcctgct gaataacttc 480tatcccagag aggccaaagt acagtggaag
gtggataacg ccctccaatc gggtaactcc 540caggagagtg tcacagagca
ggacagcaag gacagcacct acagcctcag cagcaccctg 600acgctgagca
aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag
660ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgttga
708142235PRTHomo sapiens 142Met Asp Met Arg Val Pro Ala Gln Leu Leu
Gly Leu Leu Leu Leu Trp1 5 10 15Leu Pro Gly Ala Arg Cys Ala Ile Gln
Leu Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser Val Gly Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser 35 40 45Gln Gly Ile Ser Ser Ala Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Lys 50 55 60Ala Pro Lys Leu Leu Ile
Tyr Asp Ala Ser Asn Leu Glu Ser Gly Val65 70 75 80Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 85 90 95Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 100 105 110Phe
Asn Ser Tyr Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 115 120
125Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
130 135 140Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe145 150 155 160Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln 165 170 175Ser Gly Asn Ser Gln Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser 180 185 190Thr Tyr Ser Leu Ser Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu 195 200 205Lys His Lys Val Tyr
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 210 215 220Pro Val Thr
Lys Ser Phe Asn Arg Gly Glu Cys225 230 235
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